Dear NASA planetary protection center of excellence (Planetary Protection) and other interested parties including the ESA: Planetary Protection and Space Debris Mitigation Officer and international organizations like the WHO and FAO

[Please forward to your point of contact for comments under NEPA]

I am following up on my public comments on your plan to return samples from Mars in the early 2030s after you requested comments as required by NEPA. The Council on Environmental Quality advises the public that if our concerns aren’t resolved during comments on the Environmental Impact Statement (EIS), we need to contact the agency:

Your first line of recourse should be with the individual that the agency has identified as being in charge of this particular process.
(CEQ, 2007, A citizen’s guide to the NEPA: Having your voice heard : 28)

[using a simple form of inline cites as a linked title to the paper, and page number etc and with this longer format I find it works better in terms of readability to put the cite at the end of a quotation rather than in the sentence preceding it, especially when titles of papers are long]

Video presentation for this intro:

Video: Open letter to NASA: let’s keep Earth 100% safe: when you return samples from Mars in the 2030s

This is my response to your draft EIS and to the final PEIS which is almost word for word identical and hasn't taken account of any of the major issues raised by the public comments. For my response to your replies to our public comments see the separate page:

• Dear NASA, your final Environmental Impact Statement (EIS) for samples returned from Mars and replies to public comments show
  - that though NASA is excellent on space science
  - your team is unfamiliar with essential basic planetary protection concepts
  - for example, your central Mars meteorite argument is rebutted by its National Academy of Sciences cite
  - this is only one of numerous basic mistakes in planetary protection in the EIS itself and in your responses to public comments
  - anyone familiar with the basic concepts in this discipline couldn't make these mistakes
  - this needs to be tackled at a higher level than your Mars Sample Return team
  - and needs a new EIS
  - NEPA doesn't require peer review but does require scientific integrity and expertise in the relevant disciplines
  [MID EDIT - LOTS OF WORK TO DO ON THIS PAGE STILL]

In the first round of public comments I raised a serious issue with use of a Biosafety Level 4 facility (BSL-4I). I also proposed a solution that keeps Earth 100% safe.

• (Walker, 2022, Comment by posted on May 16th) (with attachment that goes into more detail)

From the NEPA legislation, you have a legal requirement to identify all alternatives, information and analyses presented in the first round of comments in the draft EIS § 1502.17. You also have a legal requirement to evaluate any reasonable alternatives § 1502.14. .

The EIS doesn't present ANY alternatives except the legally required "no action" (NASA, 2023, MSR FINAL PEIS :2-24). In the following section they dismiss various other alternatives such as returning samples to the ISS or to a human operated lab on the Moon, but don't look at this one which would cost far less, protect Earth 100%, and solves all the problems with those suggestions (including the microgravity issue which it solves with artificial gravity in a centrifuge).

There was no mention in the draft EIS that anyone suggested returning samples to a miniature life detection lab above Geostationary Earth Orbit (GEO). There is still no mention of the suggestion in the final PEIS except a brief dismissal. This is all you say:

Refer to the previous responses for AL-001 and AL-002

This is a reasonable alternative since scientists say we can do in situ life detection as far away as Jupiter's moon Europa (Hand et al., 2017, Report of the Europa Lander Science Definition Team : xi).. Also, there is no mention anyone raised any issues with your proposal to use a BSL-4.

I then commented on the Draft PEIS itself. I found numerous very major mistakes. I summarize these briefly in 14 points along with a more detailed summary of my suggestion to keep Earth 100%.

• (Walker, 2022, Comment posted December 20th) (with 8 attachments that go into more detail)

Those 8 attachments include a graphical abstract of the reasonable alternative. Since then I've realized safety testing won't work even for samples returned in clean containers, at least if we want a high level of assurance to protect Earth. This is because we need to consider worst case scenarios for planetary protection, and in this case there are plausible scenarios where Mars has ultra low levels of viable microbes in locations like Jezero crater. It could have no more than a few viable microbes, imbedded in cracks in dust grains in the entire sample collection. We couldn't rule that out based on just the first samples returned from Mars.

I quote from it below with links to the attachments and description of each one.

• Summary of the 14 main points as presented in my public comments under NEPA

See below:

• We can't use safety testing either in orbit or on Earth to prove the samples are safe to release unsterilized
  - all Perseverance's samples would go to hold and critical review following the COSPAR Sample Safety Assurance Framework
  - because of the terrestrial contamination at 8.1 ppb
  - and because we don't have adequate knowledge of the terrestrial contamination sent to Mars
  - well over 1000 genera with two few reads to identify them and only 54 genera identified
  [IN NEPA BOTH]
   
• Safety testing in an orbital facility can't be used at all if microbes occur in very low concentrations of only a few microbes per sample - even if, improbably, most of the sample was destructively tested for biosignatures, we couldn’t rule out a viable spore perhaps imbedded in a crack in a dust grain in the remaining fraction of the sample

Here is a new version of that abstract

, Mars Sample Return overview infographic,

Text on graphic: Mars may be like Earth's driest deserts, with small niches for life adapted to extreme environments, perhaps only habitable at microbial scales.

Sample Cache Rover (Perseverance) - ESA Fetch rover - Mars ascent vehicle - samples have too much terrestrial contamination for astrobiology.

ESA sends small sterile containers on its lander to return salts, dirt, atmosphere, dust from its surroundings.

If possible, the ESA lander also sends a Marscopter to travel to a nearby young crater to return a small pebble with young exposure age

No risk to Earth's biosphere. - Above GEO.

Scientist say we can do in situ life detection as far away as Jupiter's moon Europa - Europa lander life detection lab

All returned samples are sterilized - humans go nowhere near the satellite

Sample retrieval lander mission (launch 2028) Earth Return Orbiter mission (launch 2028) samples returned 2033

Graphic obtained by modifying the ESA graphic, (Oldenburg , 2019, , Mars Sample Return overview infographic)

It's based on

Europa Lander life detection lab

NASA;s idea for Jupiter's moon Europa

Europa lander: total mass 42.5 kg

My suggestion: return samples to a miniature lab like this (adds centrifuge for artificial gravity)

But above GEO

Keeps Earth 100% safe from Mars microbes

Graphic from (NASA, 2017, Europa Lander Study 2016 Report)

And the satellite itself with the centrifuge:

Text on graphic: Bonus samples in STERILE containers returned to satellite perhaps 50,000 or 100,000 km above GEO in what would be Earth’s ring plane if it had a ring system.

• NOT for safety testing
• Returned for astrobiological study – nexus of expanding off-planet astrobiology lab.
• Minimal forward contamination.
• Humans nowhere near this.
• Centrifuge to replicate martian gravity.

Many instruments placed in centrifuge along with the dust and operated remotely from Earth. Illustration shows:

• Chiral labeled release.
• SETG from sample acquisition through to DNA sequence all automated in 2 units, each can be held in palm of hand.
• Astrobionibbler microfluidics can detect a single amino acid in a gram of sample

This would be minimal cost for NASA as the instruments would be funded by universities.

Graphic shows: (NOAA’s new GOES-17 weather satellite has degraded vision at night) just to have an image of a geostationary satellite, not that it would be a $2.5 billion dollar satellite. SETG from (Mojarro et al., 2016, SETG: nucleic acid extraction and sequencing for in situ life detection on Mars). Astrobionibbler from (Elleman, 2014, Path to Discovery) ISS centrifugal motor for plant experiments, dialable to any level from microgravity to 2g (Centrifuge Rotor [biology experiment on the ISS])

It would use a very safe orbit in the inclined Laplace plane above GEO.

• We can use a very safe orbit in the inclined Laplace plane far above GEO
  - where Earth's ring particles would orbit if it had a ring system
  – about as far as you can get in delta v from Earth or the Moon in cislunar space
  - and in the same plane as a proposed disposal orbit for geostationary satellites
  - even high area to mass ratio debris (HAMR) from the new satellite such as insulation debris can't contaminate satellites in GEO

Since I submitted that last comment, I’ve been working on a preprint and literature survey to look at this in more depth, based on a paper I was already working on, on planetary protection for your sample return mission before you started on the EIS process.

I did this because there is no up to date Mars sample return study. The ESF study in 2012 focuses mainly on the size limit and level of assurance (Ammann et al., 2012, Mars Sample Return backward contamination–Strategic advice and requirements) and is itself a decade out of date now. The last major Mars sample return review was in 2009 (SSB, 2009, Assessment of planetary protection requirements for Mars sample return missions). The previous major study was 12 years earlier in 1997 (SSB, 1997, Mars Sample Return: Issues and Recommendations (1997) ). The 2009 study was published just weeks before the unexpected discovery of droplets of salty water on the legs of the Phoenix lander which were later replicated in Mars simulation chamber experiments (Rennó et al., 2009, Possible physical and thermodynamical evidence for liquid water at the Phoenix landing site).

There have been many other surprises since then. See below:

• ALL four of the currently confirmed or uncertain novel microenvironments found on Mars relevant to native Martian life were surprises
• Predictions from the literature for seven potential microenvironments we can't expect to spot from orbit

My literature survey turned up many other topics that would need to be covered in a new Mars Sample Return review similar to the Space Studies Board review of 2009. See

• Topics that need to be covered in a future Mars sample return backwards contamination study (not likely to be a complete list)

This didn't change my conclusions and suggestions in the last comment but it added more depth to them and means they can be grounded in the most up to date science. I will add [NEW] to section titles here for material that turned up as a result of the literature survey. Everything else is already covered in the 8 attachments to my final comment (Walker, 2022, Comment posted December 20th). Most are already covered in the attachment to my response during the first round of public comments in May.(Walker, 2022, Comment posted on May 16th)

I emailed your point of contact Dr Alvin L,. Smith II on 18th December as recommended by the CEQ. I got no response. I am trying again now that you have released your final PEIS. I look forward very much to your reply.

I will also share this with the ESA Planetary Protection Office. The European Space Foundation study in 2012 agrees with all other major Mars sample return studies that the potential for large scale effects is not demonstrably zero. It is also the most recent Mars sample return study. This means that ESA also have responsibilities under various laws that apply to ESA member countries including the EU directive 2001/42/EC which requires an independent legal process similar to NEPA . The conclusions of the ESF study also mean that this mission is relevant to international organizations like the WHO and the FAO.

NASA has a legal requirement under NEPA to discuss all major points of view on environmental effects. See § 1502.9. This view NASA omitted is not only a major point of view on the potential for environmental effects; it is the consensus of ALL the major peer reviewed studies of a Mars sample return mission. Including the view of the European Space Foundation that the Mars samples should be treated as the most hazardous Earth organisms known, also shared by your former planetary protection officer Cassie Conley. That video is even still online as a NASA official video.

See below:

• ALL major studies to date through to the European Space Foundation study in 2012 that say the potential for even large-scale effects is NOT demonstrably zero
  - experts do believe the risk of large-scale harm to human health or the environment is low
  - but even a low risk of this nature is HIGHLY SIGNIFICANT
   
• 2012 ESF study says Mars samples should be treated as risk group 4 Earth organisms with high individual and community risk, usually no treatment available, until we know more
  - and in an official NASA video your former planetary protection officer Cassie Conley says we will treat them as the most hazardous organisms known
   
• Based on what the European Space Foundation Sample Return study of 2012 says about a low risk of large-scale effects
  - this mission also has international implications for ESA member countries, and the EU and also internationally
  - including obligations under EU Directive 2001/42/EC, the Espoo convention and the Biodiversity Convention

The draft EIS never mentions that the samples have to be contained as if they were the most hazardous organisms known. All you say is that

The relatively low probability of an inadvertent reentry combined with the assessment that samples are unlikely to pose a risk of significant ecological impact or other significant harmful effects support the judgement that the potential environmental impacts would not be significant. https://www.regulations.gov/document/NASA-2022-0002-0176

(NASA, 2023, MSR DRAFT PEIS :3-16)

[Same wording in the final PEIS (NASA, 2023, MSR FINAL PEIS :3-16))

You get to this conclusion as a result of using four invalid arguments widely believed in the space community, especially the Mars meteorite argument.

• These four plausible but invalid arguments reinforce false beliefs in the space community - which they use in good faith to recommend dropping all planetary protection for Mars and Earth

The EIS does mention the National Academy of Sciences sample return study from 2009 and also from 2019 but doesn't summarize the conclusions correctly suggesting incorrectly that there are no other major views on the environmental effects to be considered.

• The final Environmental Impact Statement covers the NEPA requirement to consider all major points of view on environmental effects with a statement that the National Academy of Sciences Mars sample return study in 2009 and Martian moons sample return study in 2019 "affirmed the consensus" that the Mars meteorite argument is valid
  - BOTH STUDIES said clearly this argument is INVALID
  - no mention of Cassie Conley's statement in an official NASA video that the samples will be treated as the most hazardous Earth organisms known
  - no mention of the European Space Foundation recommendation to treat the samples as risk group 4 organisms

I help scared people over the internet, and since this is an open letter, before I go any further, it is very important to me to explain clearly early on that the risk is likely low and especially for this mission. I think Dr Margaret Race's analogy of a smoke detector summarizes the consensus of the sample return studies perfectly. (Rummel et al., 2000, Opinion: No Threat? No Way : 5)

Hand installing smoke detector labeled “NASA” and wooden ceiling of a house labeled“Earth”

(Smoke detector graphic from The EnergySmart Academy)

I am doing all this to get NASA to install a smoke detector but one that I think is exceptionally important despite the low risk, because Earth’s biosphere is a “house” with billions of people in it.

For more about how this is about a likely very low risk but a very low risk of high consequence for the worst case scenarios, like house fires and smoke detectors see this separate page:

• Why I’m asking NASA to re-examine plans for its Mars sample return mission
  - like asking an architect to install a smoke detector
  - remote chance of house fire
  - but a house of billions of people
  - and at some point for sure they will have to do it

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NASA has been world leading in planetary protection - but since early this century it moved the other direction against the advice of the Space Studies Board [NEW]

Video: Open letter to NASA: Your EIS has MAJOR planetary protection mistakes - only best case scenarios

[At the time I wrote the public comments I wasn't aware of the reason for all the mistakes]

NASA has been world-leading in protection of Mars from forward contamination – from any terrestrial life that might get there and proliferate in any habitats that might exist in the Martian desert landscape.

You have also been world-leading in your literature on a Mars sample return. I was so surprised at the mistakes I found in the EIS which I listed in my response on the last day of public comments which you have surely read by now (Walker, 2022, Comment posted December 20th).

However I understand better now. I found out you closed down your planetary protection office, which operated from 1997 to 2017 (Voosen, 2017, With planetary protection office up for grabs, scientists rail against limits to Mars exploration)..

Then from the brief history of what happened by the Space Studies Board in 2018 (Space Studies Board, 2018, Review and Assessment of Planetary Protection Policy Development Process : 26), before then you closed down the interagency panel in 2006 which could have advised you, for instance on public health, and issues with lab safety and quarantine. By its charter (Planetary Protection Advisory Committee Charter) this panel included scientists from:

• Department of Agriculture
• Department of Energy
• Department of Health and Human Services
  • National Institutes of Health
  • Centers for Disease Control and Prevention
• Department of Interior
• Department of Transportation
• Environmental Protection Agency
• National Science Foundation
• Executive Office of the President

It also had at least four members knowledgeable in one or more of the fields of bioethics, law, public attitudes and the communication of science, the Earth’s environment, or related fields.

Then you closed down the planetary protection subcommittee in 2016 (Space Studies Board,, 2018, Review and Assessment of Planetary Protection … : 26).

It seems systemic, not an individual decision, simply that NASA's mission planners and engineers collectively paid less and less attention to the other agencies and to the planetary protection advisors, until they no longer served any useful function. As I understand it, that came first before the decision to close them down.

As the Space Studies Board describe what happened,

After a NASA task force reiterated the need for this type of committee, the Planetary Protection Advisory Committee (PPAC) was formed in 2000 under the aegis of the NASA Advisory Council (NAC).
...
PPAC’s charge was to advise the OPP [Office of Planetary Protection], review proposed missions at an early stage, and assign a planetary protection category to them. The OPP then informed the mission team as to the necessary planetary protection requirements to be met.

The first change was in 2006 when the NASA Advisory Council (NAC) was re-organized removing all the members of the interagency panel (PPAC) including the chair so they had no representatives at the highest level. Instead it became the Planetary Protection Subcommittee - a subcommittee that reported to a science committee that in turn reported to NAC (Space Studies Board, 2018, Review and Assessment of Planetary Protection … : 26). .

During this period, neither the PPAC nor the Planetary Protection Subcommittee received the attention needed for renewing and refreshing the membership. This problem was particularly evident in the reduced participation of federal agencies and other national space agencies. By 2016, the committee had become completely moribund, and it was formally disbanded in late 2017.

(Space Studies Board, 2018, Review and Assessment of Planetary Protection … : 26. 27).

This seems to be a record of their last recommendations before you closed down the planetary protection subcommittee. Your planetary protection experts were advising you of the need to ensure that the sample collection process on Mars was clean. (NAC Science Committee, July 25-27, 2016: 9)..

As best I understand from the materials available in those cites, instead of fixing this issue with terrestrial contamination identified by the PPS, your team ignored their recommendations and you then closed the subcommittee. In this way you simplified the engineering challenges for this mission, but this decision has serious consequences for the astrobiological interest of the mission as currently conceived. John Rummel put it like this, as reported by Paul Voosen for Science:

If the issues aren't resolved, Rummel says, the rover could be headed for a bureaucratic "train wreck".

(Voosen, 2017, With planetary protection office up for grabs, scientists rail against limits to Mars exploration)

It was possible to achieve far cleaner samples but the engineers worried it might introduce a mission critical failure point.

• Engineers worried that a sterile sample collection system would have to be transported to Mars in a separate bag
  - which might not open - a potential mission critical vulnerability

NASA did keep to the limits set by the Organic Contamination Panel's in 2014, but these were high levels with their main motivation to be able to do a preliminary survey of the organics which can detect the organics in our Mars meteories. However our meteorites come from at least several meters below the surface and the youngest of them left Mars only a few hundred thousand years ago (700,000 years by crater count for Zunil crater). So their organics are far less degraded by ionizing radiation than typical surface rocks on Mars.

The Organic Contamination Panel's limits were a compromise and weren't informed by the very low levels that astrobiologists wish to look for for past life, and it wasn't informed by levels actually found on Mars because Viking, Phoenix and Curiosity all used pyrolisis (heating until the organics vaporize, without oxygen). This breaks any organics up into small pieces.

• The origins of NASA's 10 ppb permitted contamination per biosignature
  - their Organic Contamination Panel in 2014
  - based on a 10 to 1 signal to noise ratio for measurements of Martian meteorites for the most abundant amino acids
  - however the martian meteorites were ejected from meters below the surface of Mars
  - and also had less oxidants than we expect from surface rocks
  - they did take account of ionizing radiation
  - but didn't take account of the effect of ionizing radiation on surface rocks in the presence of oxidants

Recent research has found the organics to be especially vulnerable to ionizing radiation in the presence of the surface oxidants.

• The Perseverance samples will almost certainly be valueless for past life searches (and likely for present-day life too)
  - 8.1 ppb of terrestrial organics seems low compared to 100 ppb until you realize that the ionizing radiation leaves < 0.1 ppb of recognizable organics
  - astrobiologists want to send instruments to Mars able to detect a single amino acid in a gram
  - [IN NEPA BOTH - the < 0.1 ppb figure is NEW]

See below:

• Planetary protection officer John Rummel's image of a train wreck for the permitted levels of terrestrial contamination of the samples
  - one of the last things your planetary protection experts warned you about before you closed down the planetary protection subcommittee
  - risk of returning samples of no astrobiological interest [NEW]

and:

• What we need to prevent in 2033: NASA like the Titanic headed for an iceberg
  - when scientists and the public realize NASA has no scientifically credible plans to protect Earth
  - and has permitted levels of terrestrial contamination that likely make its samples of virtually no interest for astrobiology past or present

This is why I suggested bonus samples collected in clean containers in my final comment on the draft EIS. These could be sent with the ESA fetch rover together with a sterile scoop to pick up the dirt. A thimbleful of dirt (preferably containing salts), dust collected in a clean air filter, a clean sample of atmosphere and an uncontaminated pebble from a recently excavated crater could transform this mission in terms of astrobiological interest (Walker, 2022, Comment posted December 20th).

Pavlov et al suggested looking for recent impact craters and rocks exposed by rapid wind erosion or perhaps in a deep valley.
(Pavlov et al., 2022, Rapid Radiolytic Degradation of Amino Acids in the Martian Shallow Subsurface: Implications for the Search for Extinct Life : page 113)

As I understand it, this seems systemic, not an individual decision, simply that NASA's mission planners and engineers were no longer listening to the other agencies or the planetary protection subcommittee, so they were no longer functioning. That seems to have come first before the decision to close them down.

The central issue here is, NASA:

• is very expert on launching spacecraft and missions to other planets.
• is NOT expert on public health or the environment.
• was expert on planetary protection
• no longer has anyone on the team with the relevant expertise on planetary protection for a Mars sample return.

This doesn't seem to be a result of any particular decision by any scientist or administrator but rather a systemic issue to do with how the whole organization works. As best I understand it based on the history, NASA

• is an organization that works very well for launching missions into space but it is not set up for this type of a task.
• Rather than adapt, it's moved the other way and has cut off communications with other agencies and its former planetary protection officers who said they are mistaken.
• needs to find a way to turn this around and adapt - not an easy thing for a big organization to do

Your current planetary protection engineer says your priority for planetary protection now is to prepare the way for humans to go to Mars as fast as possible, and you want other agencies to help support this goal. He is now an employee in your Office of Safety and Mission Assurance, with no independence from NAS. This is what he says:

This isn’t the Planetary Protection of the past — we are doing things differently. We have a different approach and philosophy.

There’s still a lot of work to go as we start to pave the way to humans on Mars — we’ve never done that, it’s a new precedent, so we’ll need that continued support to help with managing those knowledge gaps, including management support, engineering support and of course funding support.

The rubber is hitting the road; it’s time to get it done and we need that collective agency support to do that.

(NASA, 2023, SMA Leadership Profile: Nick Benardini)

So, NASA wants the collective support of other agencies - but it's closed down the interagency panel which could have alerted them to issues with their plans that might concern other agencies.

You made these decisions against the repeated recommendations of the Space Studies board of the need for external peer review of your plans [NEW]

This is against the repeated advice of the Space Studies Board that you need such a panel. This was the conclusion of the 2009 Sample Return study written three years after you closed the interagency panel:

It is clear to the committee that NASA will need to obtain continuing interagency advice (e.g. from the Centers for Disease Control and Prevention and relevant biosecurity agencies and organizations) on planetary protection policies and compliance, similar to the functional role played by the Interagency Committee on Back Contamination (ICBC) during the Apollo program. At present, important advice is provided via the interagency representation on NASA's internal Planetary Protection Subcommittee. However PPS currently reports via the Science Committee of the NASA Advisory Council, an arrangement that arguably leads to conflicts of interest with science and mission efforts .

(Assessment of planetary protection requirements for Mars sample return missions : 67 - 68)

The Space Studies Board repeated this most recently in 2018 saying that it is needed for peer review (amongst other reasons):

Finding: The development and implementation of planetary protection policy at NASA has benefited in the past from a formally constituted independent advisory process and body. As this report is written, both the advisory body and process are in a state of suspension.

Recommendation 3.6: NASA should reestablish an independent and appropriate advisory body and process to help guide formulation and implementation of planetary protection adequate to serve the best interests of the public, the NASA program, and the variety of new entrants that may become active

The roles of the advisory body include the following:

[other roles] …

Act as a peer review forum to facilitate the effectiveness of NASA’s planetary protection activities.

(Space Studies Board, 2018, Review and Assessment of Planetary Protection … : 61 - 62)

This wasn’t done.

This leaves public comments like mine as the only method you have left to spot your numerous mistakes in the EIS - which arose because you no longer have anyone left in the team trained in planetary protection - this is the time when you most need peer review [NEW]

In this way NASA effectively shut down all external and internal critical peer review of your plans at a time when you most need it to guard against such mistakes.

This leaves public comments like mine as your only remaining way to find planetary protection mistakes in your sample return plans.

We have these public comments only because you are legally required under NEPA to request these comments, and to respond to any major issues raised during the process. See Planetary protection issues abstract and NASA's legal requirements under NEPA

The Environmental Impact Statement uses many invalid arguments,, widely believed in the space community but rebutted long ago, building up to the conclusion that any environmental effects would not be significant - this shows that you had nobody on the team with extensive familiarity with the literature on planetary protection for a Mars sample return [IN 2ND NEPA]

Your EIS has several arguments that lead you to the conclusion that any environmental effects would not be significant. If that was true there would be no need for any planetary protection. However these are old arguments that were rebutted long ago.

From a planetary protection point of view, this is arguably the most important sentence in the draft EIS, where you argue that if there is life on Mars it has already got here

“The natural delivery of Mars materials [i.e. martian meteorites that reach Earth] can provide better protection and faster transit than the current MSR mission concept … First,

First, potential Mars microbes would be expected to survive ejection forces and pressure (National Academies of Sciences, Engineering, and Medicine and the European Science Foundation 2019) [CITE REBUTS SENTENCE], …”

(NASA, 2023, MSR FINAL PEIS 3–3),

[my comment in red and emphasis added to highlight the discrepancy]

this argument is rebutted by the very cite you attach to the sentence.

“The sample may well come from an environment that mechanically cannot become a Mars meteorite. The microbes may NOT be able to survive impact ejection and transport through space.”

(SSB, 2019, Planetary protection classification of sample return missions from the Martian moons : 45)
[CAPS added to highlight the discrepancy]

As your cite also says in that quote, some of your samples come from materials that can't mechanically become a Mars meteorite such as the dust, dirt and salts. In a scenario with native martian life it could have organisms such as photosynthetic life that is limited to those surface habitats and can't grow deep underground. See below

• Your own cite for the Mars meteorite argument rebuts the sentence it's attached to
  - it says samples may come from an environment (such as surface dust, dirt, and salts) that can’t physically become a meteorite
  - it says "microbes may NOT be able to survive impact ejection or transport through space"

This Mars meteorite argument is widely believed in the space community, and if it was valid we could drop all planetary protection of samples returned from anywhere on Mars. Few except those familiar with the planetary protection literature seem to know that it was shown to be invalid by the Space Studies Board in 2009

… Thus it is not appropriate to argue that the existence of martian meteorites on Earth negate the need to treat as potentially hazardous any samples returned from Mars by robotic spacecraft.

(SSB, 2009, Assessment of planetary protection requirements for Mars sample return missions : 48).

See below:

• Microbes from Jezero crater do NOT get to Earth better protected and faster in Mars meteorites

Mileikowsky et al. who first established the potential for microbes to travel from Mars to Earth, say most microorganisms known wouldn’t be able to travel through space:

Whereas this harsh environment sets a definite barrier for most microorganisms known, some have developed survival strategies, by transforming into a dry state, the so-called anhydrobiosis ...,

(Mileikowsky et al., 2000, Natural transfer of viable microbes in space: 1. From Mars to Earth and Earth to Mars : 392).w

Also our Mars meteorites left Mars at least hundreds of thousands of years ago and come from at least 3 meters below the surface, and by impact modeling, probably from 50 meters or more below the surface. See below:

• (MORE DETAILS - BECAUSE MANY BELIEVE THIS INVALID ARGUMENT)
  our martian meteorites left Mars at least hundreds of thousands of years ago
  - all come from at least 3 meters below the surface
  - modeling suggests they all came from a depth of at least 50 meters
  - many microbes that live in rocks need access to sunlight
  - also many microbes are not adapted to live inside rocks
  - also most of the martian subsurface is very uninhabitable
  - uniform -73°C at a depth of 12 centimeters or deeper
  - except at geothermal hot spots if any

That you use this argument shows that there is nobody in a position to review the EIS who is thoroughly familiar with the planetary protection literature for a Mars sample return.

For another example:

Existing credible evidence suggests that conditions on Mars have not been amenable to supporting life as we know it for millions of years (… National Research Council 2022).
(NASA, 2023, Mars Sample Return FINAL PEIS : 1–6)

Your very cite for this "existing credible evidence" talks about exploration to help establish if there are localized habitable regions on Mars!

“The exploration of … Mars … will help establish whether localised habitable regions currently exist within these seemingly uninhabitable worlds”. (Origins, Worlds, and Life : 393 [Click on the X to go straight to page 393]).

Also if we look at NASA's Mars science goals, the second half of Goal 1 is to determine if Mars still supports life

Goal 1: determine if Mars ever supported, or still supports life

(Mars science goals, objectives, investigations, and priorities: 2020 version : 9).

And your iMOST team recommended they test Perseverance's samples for present day life:

• test these very samples for metabolism and respiration (Beaty et al., 2018, iMOST : 92).
• experiment to see if they can get anything to grow from them (Beaty et al., 2018, iMOST : 93).

See below:

• No we don't have evidence Mars is uninhabitable - you even plan to test Perseverance's samples to see if any lifeforms detected from Mars are still alive
   
• Your own cite for “existing credible evidence" that Mars is uninhabitable is actually about searches to see if "habitable regions currently exist " on Mars

Is it possible the authors of the EIS were not familiar with NASA's own Mars science goals?

Or - perhaps instead it is due to a lack of familiarity with how to do risk assurance? All the way through the EIS the assessment of risk is to do with looking at the best cases. Here the best case is that Mars is uninhabitable because that will mean there is no need for any planetary protection.

There are other major mistakes of this sort in the draft EIS always in the direction of evaluating the planetary protection risk as non existent by looking at best case scenarios for planetary protection. In some cases it may be just lack of familiarity with the literature.

All of the most important sentences for planetary protection have similar issues. I go into this in more detail in the sections below, starting with:

• The errors I found (which I'm about to describe) are neophyte errors in planetary protection
  - your former planetary protection officers would have been incapable of such mistakes

Another example like this is the use of SR-SAG2 without citing the Space Studies Board review of it. The draft EIS singles out Jezero crater as a place on the Martian surface which they say is especially inhospitable for life to survive

Consensus opinion within the astrobiology scientific community supports a conclusion that the Martian surface is too inhospitable for life to survive there today, particularly at the location and shallow depth (6.4 centimeters [2.5 inches]) being sampled by the Perseverance rover in Jezero Crater, which was chosen as the sampling area because it could have had the right conditions to support life in the ancient past, billions of years ago (Rummel et al. 2014, Grant et al. 2018).

[Rummel et al., 2014 is often referred to as SR-SAG2]

(NASA, 2023, MSR FINAL PEIS :S-4)

SR-SAG2 seems to be far better known than the Space Studies Board review of it. SR-SAG2 has 308 cites in Google Scholar. The Space Studies Board review of it has only 16 cites in Google scholar. So it's an understandable omission for authors without in depth knowledge of the planetary protection literature. However it is a serious omission since the Space Studies Board paints a picture of a potentially far more habitable Mars and NASA and ESA commissioned this review partly out of a perception in some circles that the authors of SR-SAG2 were too closely aligned with the Mars Program Office. See below:

• Jezero crater may be more habitable to Martian life than NASA's cite SR-SAG2 suggests - NASA and ESA commissioned a Space Studies Board review of it out of a perception in some circles that the authors of SR-SAG2 were too closely aligned with NASA's Mars Program Office - the review found many knowledge gaps SR-SAG2 didn't address directly relevant to Jezero crater

The biological safety report follows a similar approach to risk assurance of focusing on best case scenarios. See below:

• The biological safety report similarly looks only at best cases for planetary protection - a systemic issue - needs someone on the team trained to search carefully for worst case scenarios as they miss several counterexamples that aren't hard to find

This is an example. After looking at many diseases that ARE adapted to humans, the biological safety report concludes that there is a near-zero probability of anything on Mars that could be a disease of humans (direct pathogen).

“Since any putative Martian microorganism would not have experienced long-term evolutionary contact with humans (or other Earth host), the presence of a direct pathogen on Mars is likely to have a near-zero probability.”

(Craven et al., 2021, Biological safety : 6)

However there are many examples in the literature on planetary protection that show that this reasoning is invalid. See below:

• In worst case scenarios microbes do NOT need to have long-term evolutionary contact with Earth hosts to harm us - example of tetanus which kills thousands of unvaccinated newborns every year and legionnaires disease which is a disease of protozoa and biofilms not adapted to invade lungs which also kills

Similarly they use the invalid reasoning that life on Mars wouldn't be able to survive on Earth, because it would be too different, with the wrong environmental and nutritional conditions. That may well be the case for SOME scenarios for species of life on Mars but it's not valid to reason that in ALL scenarios ALL martian species would be unable to survive on Earth.

“There are many described extremophiles that may survive in environments that are extreme to human or animal life (e.g. extremes of temperature or pressure) but do not survive under conditions in our normal habitat (Merino et al. 2019) … Thus, it is plausible that any Martian microbe, after it arrives on Earth, would not be viable on Earth due to a lack of its required Martian nutritional and environmental conditions.”

(Craven et al., 2021, Biological safety : 6)

This is shown to be invalid even by their own cite, Merino et al., Its table of extremophiles include one species isolated from Canadian permafrost that can grow up to human blood temperature. See below:

• In worst case scenarios microbes that live in extreme conditions on Mars would be pre-adapted to warm conditions on Earth
  - the Biological Safety Report's own cite has an example of a microbe from the Canadian permafrost isolated at -16°C, may be able to grow below -25°C, with optimal growth at 25°C and able to grow up to human blood temperature

As for the required nutrients, there are many Mars analogue organisms that would be able to survive on Mars if there is a source of water available to them and also survive on Mars. For one of them see below::

• Detailed illustrative mirror life scenario - an analogue of the blue-green algae chroococcidiopsis
  - one of our best candidates for a Mars analogue organism
  - can find all its nutritional requirements in basalt or gabbro, also sea water, fresh water and soil with strains found almost everywhere on Earth
  - and a mirror life analogue on Mars would likely be adapted to metabolize normal organics because of infall from space

This is the first of my two illustrative scenarios to encourage space agencies to pay more attention to their responsibilities for planetary protection for Earth's biosphere, see next section.

All these plausible but invalid arguments build up to the invalid conclusion in this draft EIS that flies in the face of all previous planetary protection studies for a Mars sample return:

The relatively low probability of an inadvertent reentry combined with the assessment that samples are unlikely to pose a risk of significant ecological impact or other significant harmful effects support the judgement that the potential environmental impacts would not be significant.

(NASA, 2023, MSR FINAL PEIS :3-16)

Two illustrative scenarios developed to encourage space agencies to pay careful attention to their planetary protection responsibilities
- independently evolved life based on mirror organics [IN NEPA BOTH]
- and a novel fungal genus from Mars similar to Aspergillus fumigatus [IN NEPA BOTH for fungal pathogens, specific Aspergillus analogue is new]

I developed two illustrative scenarios with the aim to motivate space agencies to take their planetary protection responsibilities seriously. These are not intended as high risk examples, but rather to show that just as for the house fire example, we have to look at worst case scenarios even if they appear to be very low risk.

Under NEPA this is covered in section § 1502.21 (c) and (d)

(c) If the information relevant to reasonably foreseeable significant adverse impacts cannot be obtained because the overall costs of obtaining it are unreasonable or the means to obtain it are not known, the agency shall include within the environmental impact statement:

(1) A statement that such information is incomplete or unavailable;

(2) A statement of the relevance of the incomplete or unavailable information to evaluating reasonably foreseeable significant adverse impacts on the human environment;

(3) A summary of existing credible scientific evidence that is relevant to evaluating the reasonably foreseeable significant adverse impacts on the human environment; and

(4) The agency's evaluation of such impacts based upon theoretical approaches or research methods generally accepted in the scientific community.

(d) For the purposes of this section, “reasonably foreseeable” includes impacts that have catastrophic consequences, even if their probability of occurrence is low, provided that the analysis of the impacts is supported by credible scientific evidence, is not based on pure conjecture, and is within the rule of reason.

My two scenarios are, for environmental effects: See below:

• However we need to take great care with bonus samples too
  - Mars could have astrobiological surprises as astonishing as the carbon dioxide geysers were for geology
  - like mirror life

And for human health and wildlife: 

• Illustrative worst case scenario for human health and wildlife (especially birds):
  - NOVEL GENUS OF FUNGI from Mars able to infect us similarly to Aspergillus fumigatus
  - harms 200,000 immunocompromised a year, with high fatality rate 30% to 95%
  - harms 48 million with an allergic lung disease (ABPA, 2.5% of asthma cases)
  - harms 400,000 with a serious chronic allergic lung disease
  - and not adapted to an infectious lifestyle in any organism
  [NEW detailed scenario, fungal pathogens IN NEPA BOTH]

The particular example of a novel fungal genus similar to Aspergillus is new, but I cover fungal diseases from Mars in the preprint that I was working on before the first round of public comments. The version I attached to the second round of public comments is here:

• NASA and ESA are likely to be legally required to sterilize Mars samples to protect the environment until proven safe

I developed many such scenarios, some mentioned already in the earlier daft paper and others new to the preprint. Some of them are mentioned later in this open letter in the supplementary information in my discussion below of:

• The gnarly question of our vulnerability to a completely alien biology
  - "Whether a microorganism from Mars exists and could attack us is more conjectural. If so, it might be a zoonosis [infectious disease that jumps to humans] to beat all others"
  - Joshua Lederberg (discoverer of bacterial sex and pioneer in planetary protection)

Chester Everline
- a NASA employee and a co-author of your handbook on probabilistic risk assurance
- says that he didn't find a target probability
- and that if the meteorite argument can't be established (it can't as we saw) the EIS should consider an alternative of deferred return
- not to return the samples until the risks are better understood [NEW]

[I didn't know about his comment until the comments period was over, his comment and mine were the last two you received just before the deadline]]

Chester Everline, a co-author of your handbook on probabilistic risk assurance (NASA, 2011, Probabilistic risk assessment procedures guide for NASA managers and practitioners) found that the EIS didn’t state clearly what level of risk NASA is prepared to take for Earth’s biosphere. This is the same issue we mentioned in the last section, where NASA were not prepared to give an answer to the question:

Just how low is “low likelihood”? Is NASA’s goal specification to prevent accidental release of the Mars samples 1 in a thousand? 1 in a million? 1 in a billion?

(NASA, 2023, MSR FINAL PEIS 4-8),

- NASA just said

No outcome in science and engineering processes can be predicted with 100% certainty.

[then deflect away from the question] The safety case for MSR safety is based on ...

(NASA, 2023, MSR FINAL PEIS 4-8),

[Also, this reply is not correct, you can be 100% sure of some things - if you don't return the sample from Mars you can be 100% sure it won't affect Earth's biosphere]

Chester Everline said:

A possible consequence of unsuccessful containment is an ecological catastrophe. Although such an occurrence is unlikely, NASA should at least be clear regarding what level of risk it is willing to assume (for the biosphere of the entire planet)

...

If the MSR [Mars Sample Return] Campaign can convincingly demonstrate that material returned to Earth by MSR will be subjected to more severe conditions than those transported by natural processes, then MSR poses no greater risk to Earth than we would expect from the next Mars meteorite.

[I.e. if they can convincingly demonstrate that samples are subjected to more severe conditions for life in the sample tubes than in a Mars meteorite ejected from Mars and impacting Earth]

However, if this cannot be convincingly demonstrated the MSR Campaign should seriously consider not returning samples using the technology described in the PEIS [Provisional Environmental Impact Statement] (i.e., transition to a deferred return campaign option).
[THIS ARGUMENT WAS SHOWN TO BE INVALID MANY YEARS AGO (SSB, 2009, Assessment of planetary protection requirements for Mars sample return missions : 47)]

A better statement of options should include the possibility of delaying the return of Mars samples until the risks associated with their return are better understood

(Chester, 2022, Comment posted December 20th)

As we saw, the meteorite argument he mentions there was actually shown to be invalid by the study by the Space Studies Board in 2009, and it is also rebutted by the very cite the EIS uses for the sentence. See below:

• Microbes from Jezero crater do NOT get to Earth better protected and faster in Mars meteorites

His proposal is a deferred sample return and use of in situ missions on Mars until we know enough about life on Mars to return the samples safely.

I cover his proposal in depth in my Response to final PEIS page here:

• Your employee Chester Everline
  - one of your experts on risk assurance and co-author of your own handbook on the topic,
  - suggested another reasonable alternative in his public comment on the EIS
  - to search for life in situ on Mars first and return the samples only once any risk is well understood
  - your response to him is mistaken in a fundamental way as you reply "no outcome in science and engineering processes can be predicted with 100% certainty"
  - in situ study on Mars achieves 100% certainty that Earth is not harmed since no material is returned to Earth
  - this shows a lack of understanding of risk assurance in your response to a risk assurance expert!

and the following page:

• Many astrobiologists recommend in situ study in PREFERENCE to a sample return for their discipline
  - including a white paper by Bada et al submitted to the same decadal review that decided on this mission
  - in my literature study I found no papers more recent than 2002 recommending a Mars sample return first
  - the decadal review decided instead to use this old paper from 2002 as the basis for their decision
  - instead of this white paper submitted directly to them by experts
  - this 2002 paper is the reason they gave for a sample return mission rather than in situ missions
  - but even the 2002 "Safe on Mars" paper you cited said that in situ studies are better than a sample return once we have the capabilities (which we now have)
  - the issue is life on Mars is likely to be hard to detect and require examining a lot of material before we find our first living cell or our first clear evidence of past life
  - this is far easier with instruments on Mars where the material to study is essentially unlimited as Chester Everline put it in his comment
  - so Everline was correct and you are mistaken in your response to him with no awareness of many papers supporting his suggestion

Chester Everline doesn't suggest the idea of sterilization - but that is another way to protect Earth 100% as an alternative to a deferred sample return.

I am not able to liaise with employees of NASA who find issues with your EIS
- and it seems unlikely I can get help from former planetary protection officers of ESA or NASA
- but your first planetary protection officer John Rummel did assure me that he is convinced somebody will listen [NEW]

I sent an email to Chester Everline to ask if we could liaise– combining his expertise in probabilistic risk assurance and my familiarity with the planetary protection literature – but I was not surprised when he responded saying as a NASA employee he couldn’t engage.

I also contacted your first planetary protection officer John Rummel. He was the obvious academic to contact as principle author, co-author, or contributor to nearly all the major studies on a Mars sample return, including the 2009 Mars sample return study I just mentioned which rebutted the meteorite argument - and indeed much of the literature on the topic for the last several decades. He just said he has retired and I should contact the planetary protection office (which has not replied).

As for ESA – all this is too recent to be mentioned in the 2018 Space Studies Board report, but it seems that ESA is following NASA’s lead. First, as partners with NASA on this mission, one would expect them to sign off on the basic mission plan that is a basis for the EIS at a high level. After all, the plan is that it's their spacecraft that is going to return samples from Mars to the USA (ESA Earth Return Orbiter)

As I mentioned in the introduction, though ESA plan to return the samples to the USA, in the worst case the sample can have large scale effects outside of the USA. This means member countries of ESA have responsibilities under various local laws and international treaties. For instance they are all signatories of the Biodiversity convention which has obligations about preventing the spread of invasive species, and EU member countries have obligations under EU directive 2001 / 42/EC which is similar to NEPA.See below:

• Based on what the European Space Foundation Sample Return study of 2012 says about a low risk of large-scale effects
  - this mission also has international implications for ESA member countries, and the EU and also internationally
  - including obligations under EU Directive 2001/42/EC, the Espoo convention and the Biodiversity Convention

ESA’s planetary protection officer Gerhard Kminek is still listed as ESA’s Planetary Protection Officer on their website (Planetary protection). However he no longer has the job.

The new post was advertised as "ESA Planetary Protection and Space Debris Mitigation officer" (ESA: Planetary Protection and Space Debris Mitigation Officer) . I haven’t yet found out who got it.

The new officer has many responsibilities, including:

• Planetary Protection;
• Space Debris Mitigation;
• Nuclear Safety for applications in space;
• Re-entry safety;
• Human Spaceflight Safety.

(Planetary Protection and Space Debris Mitigation Officer)

That is way too much for one person unless they give only a small amount of attention to each job. So it looks as though ESA has followed the lead of NASA and has de-emphasized planetary protection At any rate I got no reply to the email ESA's former planetary protection officer forwarded to them.

ESA's policies for the samples returned from Mars has had much less public attention, zero attention in fact as far as I can see. I don't find any statements on the ESA website yet of any change in direction for planetary protection unlike the clear statements in the NASA website.

The ESA planetary protection page still only mentions the former planetary protection officer Gerhard Kminek. Also, there is no mention yet that they are participating in NASA's Mars sample return mission or of planetary protection for a Mars sample return so they haven't updated it to take account of this mission (ESA Planetary Protection).

According to the current plans ESA are responsible for returning the samples from Mars to Earth as described on the ESA site here: (ESA - Earth Return Orbiter) and video: How will Airbus bring the first samples ever from planet Mars - Introducing the Earth Return Orbiter

ESA has a history of careful planetary protection and haven't announced a change of policy, so I hope this open letter will encourage you to continue to take your planetary protection responsibilities seriously.

My aim is to help make this an even better mission - and we now have the technological capability to achieve 100% planetary protection for Earth - and for Mars too [IN NEPA BOTH]

My focus here is to chart a way forward for NASA to recover from these mistakes and retain its leading role in planetary protection and to do a mission with enhanced science return and safe for Earth. As I said in the conclusion to my comment:

Let's make this an even better mission and SAFE for Earth.

(Walker, 2022, Comment posted December 20th)

I’ve done some extra videos for sections of this open letter for those who prefer video presentations - and which sometimes go into more depth.

As I outlined in those 14 points (Walker, 2022, Comment posted December 20th), we now have the ability to achieve 100% planetary protection both ways.

This is partly inspired by your proposal from 2016 for a miniature life detection lab on Europa (NASA, 2017, Europa Lander Study 2016 Report) which in turn is feasible because of the extraordinary miniaturization of technology over the last two decades.

Europa Lander life detection lab

NASA;s idea for Jupiter's moon Europa

Europa lander: total mass 42.5 kg

My suggestion: return samples to a miniature lab like this (adds centrifuge for artificial gravity)

But above GEO

Keeps Earth 100% safe from Mars microbes

Graphic from (NASA, 2017, Europa Lander Study 2016 Report)

Scientists assure us it is feasible to run such a lab on Europa. So, it is must be possible to devise a lab that can operate above GEO with a latency of only a quarter of a second, and within easy resupply of Earth. Already in 2016 the scientists in the team concluded:

The Europa Lander mission concept is designed to achieve ground-breaking science. The SDT (Science Definition Team) is confident that a payload matching or exceeding the requirements described herein could potentially reveal signs of life on Europa (Hand et al., 2017, Report of the Europa Lander Science Definition Team : xi).

• We can protect Earth 100% using the amazing shrinking technology of astrobiological life - detection. We will soon have the capability to make an entire telerobotic astrobiology lab only a few meters across, small enough to study the samples in a safe orbit above GEO, no humans ever go near it. NASA has a major role in developing those instruments including the most challenging of all, a miniaturized end to end gene sequencer needing no maintenance, SETG (Search for Extraterrestrial Genomes) which you are in the process of getting ready to do gene sequencing in the oceans of icy moons like Europa and Enceladus, and Mars. (Carr et al., 2020, Nanopore sequencing at Mars, Europa, and microgravity conditions). The main challenge is to develop a maintenance free replacement for the organic nanopores that need to be replaced every few weeks. It would use nanogaps instead of nanopores and would even be able to do protein sequencing (Maggori, 2023, Life detection and taxonomic characterization with MinION sequencing in Mars and icy worlds analogue environments : 230). It may well be ready well before the samples return in 2033.
  
  By 2016, the Europa lander science definition team were already confident we could run such a lab as far away as Jupiter's moon Europa (Hand et al., 2017, Report of the Europa Lander Science Definition Team : xi). By 2033 we can reasonably expect to be able to miniaturize almost all the measurements your iMost team recommended to try to find living or recently dead present day Martian organisms in the samples (Beaty et al., 2017, iMOST : 91–3). Meanwhile
  
  Then we can do virtually all geology with sterilized samples, especially since the samples we return have likely already had more than a sterilizing dose of surface ionizing radiation since they were deposited - so we never need to return unsterilized samples to Earth.
  
  I go into details of some of the extraordinarily small and sensitive instruments astrobiologists have developed here: NASA already have worked on plans for a remote life detection laboratory on Europa in the report of the Europa Lander Science Definition Team - what we can do using a remotely operated lander on Europa we can do in a far easier fashion in a small telerobotic facility above GEO with a latency of a quarter of a second return trip
   

The reasoning that lead to this proposal is that first, even the best run human operated lab has to be able to respond to a lab leak according to the WHO (WHO, 2020, World Health Organization Laboratory biosafety manual, 4th edition : 87). See:

• Even the most well prepared biosafety laboratory has to have emergency procedures for lab leaks

The Apollo program used quarantine, but this was a decision made by NASA, who are not expert in epidemiology and they set up their internal interagency panel so they could override any decisions any of the other agencies made (Meltzer, 2012, When Biospheres Collide : 193).

• In the Apollo era NASA had an interagency panel
  - but all discussions were private and it was set up so no changes could be made to NASA's plans without NASA's own approval
   
• NASA used those powers to tell the astronauts to open the Apollo 11 door while still floating in the sea
  - against the objections of the National Academy of Sciences whose representative Vishniac said it would make the rest of the quarantine program pointless

Any epidemiologist would confirm that it is impossible to use quarantine to keep out a disease with symptomless carriers like Typhoid Mary (Mary Mallon: First Asymptomatic Carrier of Typhoid Fever). But what's more, it turns out that NASA's objective for the Apollo quarantine wasn't even to keep out a disease of humans. Their aim was just to try to ensure that if a disease got to Earth and started to spread amongst humans, that it would have an incubation period of at least three weeks. The idea was that this would give us more time to respond to it. Also quarantine couldn't keep out a novel fungal genus like Aspergillus or mirror life. See below:

• Apollo quarantine was only designed to protect Earth from diseases with an incubation period less than 3 weeks
  - and even for those, NASA's plan was to rush any technician or astronaut who got seriously ill to hospital as an authorized breach of quarantine

This was before NEPA. The plan was published on the day of launch of Apollo 11 and was never subject to legal review or public scrutiny (Meltzer, 2012, When Biospheres Collide : 452).

We might not have the same issues with lab leaks with a telerobotically controlled lab, as telerobots can be sterilized, unlike humans. However though that might be a feasible and safe alternative once the technology is mature - we have no experience of running such a lab. Then there are issues such as criminal damage, mistakes, accidents, and would it really contain all leaks? Also the need for sterilization when the facility is decommissioned if it contains mirror life.

That is what lead me to the suggestion of a small telerobotic facility above GEO. It eliminates all these issues with a terrestrial telerobotic lab. I then added the idea of a centrifuge such as the one used in the ISS to deal with the issue that many experiments need gravity to operate. So I hope you can understand the reasoning for my suggested alternative.

Then, again due to remarkable advances in technology, we are within reach of 100% planetary protection in the forwards direction too.

• In the future we can protect Mars and the entire solar system from life from Earth using NASA’s extraordinary Venus HOTTech (NASA, n.d., HOTTech) in development - technology for probes for Venus that can survive at 500°C for months with no cooling complete with short lived cameras (Long-lived in-Situ solar system explorer (LLISSE) Potential Contributions to solar system Exploration). This builds on a suggestion from 2018 (Automation Rover for Extreme Environments : Section 6.2) but modern technology makes it much easier and it will continue to get easier as this decade progresses. "The goal is to develop technology areas that will enable a long-lived lander that can survive at least 60 days at 500C." (NASA, n.d., HOTTech)
  
  It’s a much easier requirement for a 100% sterile lander for Mars. Engineers have to ensure every component can resist heating for a few minutes at 300°C. Heat it for a few minutes on the journey out and it is now 100% sterile and can be sent anywhere on Mars with no risk of terrestrial contamination. For 300°C we have many components already available, for instance fast electronics with 0.35 micron resolution (High temperature electronics) (Integrated High Temperature Circuits - Fraunhofer IMS) using silicon on insulator technology (Silicon On Insulator (SOI)). A reasonable first challenge would be a 100% sterile Marscopter - and very useful.
  
  For any more sensitive devices, we can also combine it with other methods of sterilization, for instance ionizing radiation combined with heat is especially sterilizing and could reduce the temperature needed, and CO2 snow works especially well for small devices (cavebots, aerobots, moles, etc spring to mind as well as small experiments). CO2 snow also removes all the remaining organics once the surface is already reasonably clean.
  
  See below: The best way to achieve Sagan's "vigorous program of unmanned exobiology" is to use 100% sterile landers and rovers - and we have the technology now to do it with HOTTech
   

• We can ensure that a subsystem, or maybe even all of the miniature telerobotic life detection lab above GEO is 100% clean by using the same HOTTech method to eliminate even small amounts of terrestrial forwards contamination of the samples, make sure every component can withstand heating to 300°C and heat it for a few minutes before it receives the samples from Mars.
  
  It would use miniature telerobotic handlers and microfluidics. Everything done inside a centrifuge to simulate Mars gravity - and we can simulate the atmosphere, and we can use sunlight brought in using optical fibers or just a window, to simulate day night cycles for an excellent Mars simulation chamber well beyond anything we can do on Earth - a more elaborate version of the BIOMEX Mars simulation experiment that was attached to the exterior of the ISS (Limits of life and the habitability of Mars: the ESA space experiment BIOMEX on the ISS).
  
  See: We can eliminate all these issues and make it a far simpler mission using a miniature telerobotic facility above GEO (below)

So - that’s at least one positive direction we can go in the future. I expand on those suggestions towards the end of this open letter and in far more detail in the preprint.

We are at decision point for our civilization - not just NASA - with two ways we can go - no planetary protection in the near future - or 100% planetary protection both ways in the near future [IN NEPA BOTH]

It is very important to get this first step right, the EIS and the first samples from Mars. We need to establish a good precedent for the future.

We have the option to

• Establish a precedent of 100% planetary protection of Earth with eventual aim of 100% protection both ways for maximum protection of Earth and Mars, OR.
   
• Establish a precedent of greatly lowered planetary protection with the aim of eliminating it in the near future to send humans to Mars as soon as possible

Understandably those focused on landing human astronauts on the Martian surface think this can happen sooner if we drop all planetary protection
- but they need to convince the rest of us their intuitions of a biologically harmless Mars are correct

If those who want human astronauts to land on Mars as soon as possible are correct that we will find no life on Mars, or only life that can mix with Earth’s biosphere with no harm in either direction, it is still far better for their objective if we don’t drop planetary protection quite yet.

If we put 100% planetary protection in place right now, we enhance science return. We will be able to send rovers to Mars without the noise of contamination by terrestrial life, and return clean samples that we can study again with no terrestrial contamination in our miniature telerobotic facility.

With clean missions to Mars and clean samples returned from Mars, we get a clearer picture of what’s on Mars far faster

In this way we get a far clearer picture of what we have on Mars far faster which means we can answer the questions that Mars colonization enthusiasts should want answers to too. They rely at present on hunches and vivid metaphors to persuade others that what they want to do is safe for Earth’s biosphere. If they are right they will be able to use science instead.

Also with 100% clean rovers we can send numerous miniature robotic explorers everywhere on Mars - with gigapixel cameras and the broadband communications back to Earth which NASA is planning to install this decade - which will be amazing assets for the Mars colonizers if that is what we do eventually.

Experts all say
- we can't do a classical probability estimate
- but in their expert opinion the risk is likely low
- of large-scale harm to human health or our biosphere
- Margaret Race uses the analogy of a smoke detector for a house fire
- a low risk but a risk of high significance - we do need smoke detectors [IN NEPA BOTH]

This runs through the planetary protection literature from the beginnings of the discipline, in the late 1950s to 60s, with Joshua Lederberg, Carl Sagan and others, onwards (Meltzer, 2012, When Biospheres Collide : 35, 420). The most recent European Space Foundation (ESF) Mars sample return study in 2012 concurs with the 2009 Mars sample review and indeed all major sample return studies to date:

The Study Group also concurs with another conclusion from the NRC reports (1997, 2009) that the potential for large-scale effects on the Earth’s biosphere by a returned Mars life form appears to be low, but is not demonstrably zero.

(Mars Sample Return backward contamination–Strategic advice and requirements : 20)

In more detail they say:

… it is not possible to estimate a probability that the sample could be harmful or harmless in the classical frequency definition of probability.

However it is possible to establish the risk as low, as a consensus of the beliefs of the experts in the field as represented by their experience.

(Mars Sample Return backward contamination–Strategic advice and requirements : 24)

I already mentioned the analogy of a smoke detector in the introduction to this open letter, and this is the link to my page to help anyone who gets easily scared:

• Why I’m asking NASA to re-examine plans for its Mars sample return mission - like asking an architect to install a smoke detector - remote chance of house fire - but a house of billions of people - and at some point for sure they will have to do it

I am a long term admirer of NASA - and I’m doing all this just to get you to install a working smoke detector

I am doing all this to get NASA to install a smoke detector but one that I think is exceptionally important despite the low risk, because Earth’s biosphere is a “house” with billions of people in it.

I have been a long term admirer of NASA. You have done so much and are doing so much by way of advancing science and space exploration, robotic and human. Most of our discoveries about Mars are the result of observations by NASA missions.

Carl Sagan: “we cannot take even a small risk with a billion lives”

Carl Sagan is one of my heroes and I have the same focus as him in this respect. Enthusiastic about space science. Keen on space exploration, including both robotic and human exploration. Watched the Apollo landings in amazement in the 1960s. Marveled at the Voyager “grand tour” of the solar system. But I also greatly value Earth’s biosphere and its inhabitants.

For me, the value of Earth's biosphere and its inhabitants is essentially infinite.

Text on graphic: Carl Sagan (pioneer in planetary protection - first paper in 1960)
[his first paper is (Biological contamination of the Moon)]

“I, myself, would love to be involved in the first manned expedition to Mars. But an exhaustive program of unmanned biological exploration of Mars is necessary first.

“The likelihood that such pathogens exist is probably small, but we cannot take even a small risk with a billion lives.”

[quote from: (Sagan, 1973, The Cosmic Connection – an Extraterrestrial Perspective)]
[I provide text captions for the graphics in this open latter for visually impaired readers]

In this EIS, I saw a box that looks like a smoke detector but without batteries and not up to the latest specs

In this analogy, I didn’t see a functioning smoke detector in this Environmental Impact Statement. I saw a box that looks like a smoke detector but doesn’t really function, doesn’t match modern design requirements for a smoke detector and has no batteries installed. We have to fix that.

This mission plan will not succeed without broad acceptance by the general public as well as the many scientists who do not belong to NASA [IN NEPA BOTH]

This is how John Rummel, NASA’s first planetary protection officer, put it in 2002:

“Broad acceptance at both lay public and scientific levels is essential to the overall success of this research effort.”

(Rummel et al., 2002, A draft test protocol for detecting possible biohazards in Martian samples returned to Earth : 99)

Mars sample return studies emphasize the need to involve the public early on, not just in the USA, but through fora open to representatives from all countries globally because negative impacts could affect countries beyond the ones involved directly in the mission. This is from the most recent ESF study in 2012:

RECOMMENDATION 3

Potential risks from an MSR are characterised by their complexity, uncertainty and ambiguity, as defined by the International Risk Governance Committee’s risk governance framework. As a consequence, civil society, the key stakeholders, the scientific community and relevant agencies’ staff should be involved in the process of risk governance as soon as possible.

In this context, transparent communication covering the accountability, the benefits, the risks and the uncertainties related to an MSR is crucial throughout the whole process. Tools to effectively interact with individual groups should be developed (e.g. a risk map).

RECOMMENDATION 4

Potential negative consequences resulting from an unintended release could be borne by a larger set of countries than those involved in the programme. It is recommended that mechanisms and fora dedicated to ethical and social issues of the risks and benefits raised by an MSR are set up at the international level and are open to representatives of all countries

(Ammann et al., 2012, Mars Sample Return backward contamination–Strategic advice and requirements : 59)

There will be intense public scrutiny of NASA’s plans as it gets nearer to the date the samples return to Earth. If you still don’t have a functioning smoke detector in this analogy you will have to fix this.

The obvious last minute fix is a bill in Congress in the early 2030s requiring you to sterilize all samples before they reach Earth - and with no on board sterilizer your spacecraft would have to fly past Earth and you retrieve the samples later for sterilization.

I am writing this to help you find a better solution.

Carl Sagan's strong focus on protecting humans and Earth's biosphere as our top priority is not unusual - out of those who commented - 49 out of 62 would likely agree with Sagan and nine specifically mentioned unprecedented harm as their concern [ANALYSIS OF PUBLIC COMMENTS NEW]

Carl Sagan’s strong focus on protecting Earth is not an unusual concern in the general public. Several dozen distinct members of the public expressed views on your EIS that suggest they would be in support of a similar approach to planetary protection that places a very high value on Earth’s biosphere.

Nine commentators specifically mention unprecedented harm and 49 out of 63 made comments that make it clear they would agree with Sagan’s view.

49 out of 62 shouldn’t be read as a percentage of the public as it is not a poll. But it does show that at least several dozen of the members of the public who were reached in the not very well publicized second round of comments had concerns similar to Sagan.

These are links to all the comments. Many are short and succinct but clear in what they intend to say. The ones that would surely agree with Sagan are highlighted in bold. The link text briefly summarizes the comment.

• stop mission, unprecedented harm – test first – study in separate module attached to ISS – test first – stop mission
• stop mission – test first – test first, unprecedented harm – Study in space or not at all, keep Earth 100% safe, our efforts to contain the samples may seem lax a generation from now –test first
• stop mission – need clarity about security measures – off topic – alternative design - keep Earth 100% safe
• unprecedented harm – stop mission, unprecedented harm – alternative design – test first – test first
• test first – unprecedented harm – test first – Test first– Don’t return unless 100% safe – or sterilize first
• Don’t return – don’t return until 100% safe – test first – test first–test first
• ISS first – test first – test first – unknown risk, test first–sterilize first
• extra precautions for EES reentry - sterilize first – sterilize first – sterilize in space station first – sterilize first
• do not return – do not return – do not return – send to Russia first– issues with disinfection of earth entry site
• test first– support EIS– study in situ or space lab or sterilize first – fully support, suggests more samples – – off topic (future missions need to be designed for reeuse)
• multiple cautious measures – support EIS – support EIS– test or sterilize first – sterilize first
• test in situ or don’t return – do not return – unprecedented harm, test first – unprecedented harm, return to space station

Those are 46 comments so far that would agree with Sagan.

Three more comments were very detailed with attachments making the same point.

Thomas Dehel quoting from an interview he did with Gill Levin, principle investigator for the first and only direct life detection experiment sent to Mars, who died shortly before the start of the EIS process

"I believe people will realize, especially after the Covid-19 catastrophe, that even if there’s only a small chance that something could be contagious and pathogenic, coming from a foreign planet, I don’t think it’s worth taking that chance….you don’t take unnecessary chances where the risk-to-benefit ratio is almost infinite.”

(Dehel, 2022, Comment posted December 13th)

Barry DiGregario quoted from an interview he did with Carl Woese when he was alive. Carl Woese is the biologist who used gene sequencing to identify the archaea, the third realm of life. As the botanist Otto Kandler put it: “He opened a door which nobody expected to exist” (The singular quest for a universal tree of life).

“When the entire biosphere hangs in the balance, it is adventuristic to the extreme to bring Martian life here. Sure, there is a chance it would do no harm; but that is not the point. Unless you can rule out the chance that it might do harm, you should not embark on such a course”

(DiGregario, 2022, Comment posted December 5th)

I have left out Chester Everline’s comment as a NASA employee and he also doesn’t present his own view on the topic. But he would certainly count as agreeing on the potential for unprecedented harm – and on the need to protect Earth in the case of uncertain risk of unprecedented harm.

A possible consequence of unsuccessful containment is an ecological catastrophe. Although such an occurrence is unlikely, NASA should at least be clear regarding what level of risk it is willing to assume (for the biosphere of the entire planet)

...

A better statement of options should include the possibility of delaying the return of Mars samples until the risks associated with their return are better understood

(Everline, 2022, Comment posted December 20th)

This suggests he also would agree with Carl Sagan at least in the case of an uncertain level of risk of large-scale harm.

See above:::

• Chester Everline
  - a NASA employee and a co-author of your handbook on probabilistic risk assurance
  - says that he didn't find a target probability
  - and that if the meteorite argument can't be established (it can't as we saw) the EIS should consider an alternative of deferred return
  - not to return the samples until the risks are better understood [NEW]

My own final comment also expresses the same view in 14 points, ending::

Let's make this an even better mission and SAFE for Earth.

(Walker, 2022, Comment posted December 20th)

For potentially scared people: Margaret Race’s analogy of a smoke alarm puts it very well - the level of risk is likely similar to the risk of a house fire - very low - but high significance so we do have to install a working and up to date smoke detector

I help easily scared people over the internet, and since this is an open letter, before I go any further, it is very important to me to explain clearly early on that the risk is likely low and especially for this mission See:

• Why I’m asking NASA to re-examine plans for its Mars sample return mission - like asking an architect to install a smoke detector - remote chance of house fire - but a house of billions of people - and at some point for sure they will have to do it

The errors I found (which I'm about to describe) are neophyte errors in planetary protection
- your former planetary protection officers would have been incapable of such mistakes [NEW]

Video: Open letter to NASA - focus on best-case planetary protection scenarios - like houses without fires

Your former planetary protection officers are world leaders in the field of planetary protection and they would never have let such mistakes through. I’d never expect a NASA EIS to have such mistakes.

These are systemic errors due to having nobody on the team trained in planetary protection or risk assurance
- not the fault of any individual scientist or engineer
- Space Studies Board finds it always has to educate committee members unfamiliar with basic planetary protection concepts

It’s important to recognize these mistakes are not the fault of any authors of the EIS. The Space Studies Board say they need to educate committee members unfamiliar with basic planetary protection concepts.

“ … with additional time being required to educate those committee members unfamiliar with basic planetary protection concepts.”

(Review and Assessment of Planetary Protection Policy Development Processes : 77)

They are clear and major mistakes.
- one pervasive mistake that for risk assurance you need to look at worst case scenarios (like a house fire)
- not best case scenarios (all the activities that don't lead to a house fire)

One basic mistake pervades the report - the EIS looks at best case scenarios for planetary protection throughout. Once the EIS finds a best case or several best cases that's the end of the analysis. Examples

But we need to look for worst case scenarios, as with the analogy of a smoke detector.

Microbes from Jezero crater do NOT get to Earth better protected and faster in Mars meteorites - a sample tube is like a miniature spaceship for a microbe [IN NEPA BOTH]

[I cover this in the first round of public comments in the sections that show that Zubrin's arguments are invalid]

Let’s look at an example right away.

“The natural delivery of Mars materials [i.e. martian meteorites on Earth] can provide better protection and faster transit than the current MSR mission concept … First, potential Mars microbes would be expected to survive ejection forces and pressure (National Academies of Sciences, …, 2019), …” (NASA, 2023, MSR FINAL PEIS 3–3),

This is saying, falsely, that any life returned from Mars in your sample tubes can get here with better protection and faster in a meteorite from Mars.

Although many in the space community think this meteorite argument has been established, it is

• NOT valid for samples from the Martian surface.
• valid for samples from small asteroids, comets and the Martian moons.

Your own cite rebuts it. See below:

• Your own cite for the Mars meteorite argument rebuts the sentence it's attached to,
  - it says samples may come from an environment (such as surface dust, dirt, and salts) that can’t physically become a meteorite
  - it says "microbes may NOT be able to survive impact ejection or transport through space"

The Mars asteroid argument was rebutted in the 2009 Mars sample return study from the Space Studies Board:

Thus, the potential hazards posed for Earth by viable organisms surviving in samples is [are] significantly greater with a Mars sample return than if the same organisms were brought to Earth via impact-mediated ejection from Mars

(SSB, 2009, Assessment of planetary protection requirements for Mars sample return missions : 47).  
[[See end of section "The survival of organisms ejected from Mars]]

The Space Studies Board panel goes on to say

… Thus it is not appropriate to argue that the existence of martian meteorites on Earth negate the need to treat as potentially hazardous any samples returned from Mars by robotic spacecraft.

(SSB, 2009, Assessment of planetary protection requirements for Mars sample return missions : 48).

Most species of terrestrial microbe die when suddenly accelerated from rest to faster than a hypervelocity bullet - and most of those that survive die of dehydration in a vacuum

Most terrestrial microbes couldn’t survive the sudden acceleration to ejection at many times the speed of a hypervelocity bullet and couldn’t survive the cold and vacuum of space (

Whereas this harsh environment sets a definite barrier for most microorganisms known, some have developed survival strategies, by transforming into a dry state, the so-called anhydrobiosis ..., or by producing spores, the dormant state of certain bacteria.

Concomitantly with their resistance to the adverse effects of drying, microorganisms in anhydrobiosis or spore stage are resistant to the effects of freezing to very low temperatures, elevated temperatures for brief periods, and the effects of ionizing. These characteristics make spores and anhydrobiotic bacteria especially prepared for coping with the extreme conditions of space

(Mileikowsky et al., 2000, Natural transfer of viable microbes in space: 1. From Mars to Earth and Earth to Mars : 392).

For some particular examples, e. coli, and most oxygen producing photosynthetic cells couldn't survive ejection into space. It depends most on the characteristics of the cell membrane / wall and other coatings. Photographs show that the cell walls rupture with the cell contents dispersed. That's a process that won't happen with the samples in the sample tubes. If the microbes form spores, or are placed within a thick cortex (such as for lichens) then they can survive more easily.

Planetary ejection requires survival of shock pressures. ,, The reported results suggest that for vegetative cells (which include most oxygenic photosynthetic organisms) shock pressures associated with ejection to escape velocity act as a strong dispersal filter. ... .

The presence of spore-forming states, emplacement in a thick cortex, etc., will increase the range of shock pressures under which oxygenic photosynthetic organisms (and other non-phototrophs) can survive the dispersal filter, increasing the chances of survival of planetary ejection.

The primary molecular basis of this dispersal filter is therefore exceeding the mechanical strength of cell walls/membranes. ...The experiments conducted show that the effects are specific to the particular characteristics of cell membrane/wall and other coatings of different organisms.

(Cockell, 2008, The Interplanetary Exchange of Photosynthesis : Ejection from the Planetary Surface)

Your own cite for the Mars meteorite argument rebuts the sentence it's attached to
- it says samples may come from an environment (such as surface dust, dirt, and salts) that can’t physically become a meteorite
- it says "microbes may NOT be able to survive impact ejection or transport through space"
- [partly NEW - didn't spot the direct refutation in this source in time for my final comment on December 20, 2022 - did spot that NASA's cite said it didn't study sterilization during impact]

Even your own cite in the EIS, though valid for samples from Phobos, says it shouldn’t be used for samples from the Mars surface, giving these very reasons. What the EIS says:

“potential Mars microbes would be expected to survive ejection forces and pressure (National Academies of Sciences, …, 2019), …”

(NASA, 2023, MSR FINAL PEIS 3–3),

Go to page 48 of your 2019 cite and you read (quoting the parts relevant to Jezero crater):

The reasoning regarding natural flux does NOT apply directly to samples returned from the Mars surface. The material will be gently sampled and returned directly to Earth.

The sample may well come from an environment that mechanically cannot become a Mars meteorite. The microbes may NOT be able to survive impact ejection and transport through space.”

...

Finding: The committee finds that the content of this report and, specifically, the recommendations in it do NOT apply to future sample return missions from Mars itself.

(SSB, 2019, Planetary protection classification of sample return missions from the Martian moons : 45)
[CAPS added to highlight the discrepancy]

In context, just before the passage quoted:

There are several reasons why Mars sample return (MSR) missions differ from those for collecting samples from Phobos and Deimos, including the following:

• ... The collected samples will be selected according to specific criteria designed to maximize the chance of sampling evidence of extant or extinct life ...
   
• The various physical processes evoked in the microbial contamination assessment (excavation by impact, collision with Phobos, sterilization, etc.) do not have to be considered for the assessment of potential microbial density for MSR, which could increase drastically the potential microbial density in comparison to the Phobos and Deimos sample...

(SSB, 2019, Planetary protection classification of sample return missions from the Martian moons : 45)

They don't mention surface salts, dust or dirt but they also mechanically can't become a Mars meteorite.

Your cite rebuts the sentence it is attached to. This is your cite for arguably the most important sentence in the EIS for planetary protection, because if the argument was valid there would be no need for any planetary protection.

We don't know capabilities of martian microbes if they exist. Most terrestrial microbes can't survive ejection as we saw in the previous section.

• Most species of terrestrial microbe die when suddenly accelerated from rest to faster than a hypervelocity bullet - and most of those that survive die of dehydration in a vacuum

In a scenario with Martian microbes some or even all species may be unable to survive transit to Earth in a meteorite. Then any life that lives only in the surface dust, dirt and salts couldn’t get here at all.

I cover this cite in (Walker, 2023, So many serious mistakes in NASA's Mars Samples Environmental Impact Statement it needs a clean restart : 18 - 20)]. However I only mention that their draft didn't study sterilization during ejection

The SterLim team did not include any sterilization during Mars ejecta formation in its analysis because such investigations were not requested in its study’s statement of work.

(SSB, 2019, Planetary protection classification of sample return missions from the Martian moons : 26)

I didn't notice at the time that it directly says than the reasoning doesn't apply to samples from the Mars surface.

This reasoning already shows the argument is invalid. However this reasoning is so strongly believed by many space enthusiasts that I feel it is necessary to go into more detail.

(MORE DETAILS - BECAUSE MANY BELIEVE THIS INVALID ARGUMENT)
our martian meteorites left Mars at least hundreds of thousands of years ago
- all come from at least 3 meters below the surface
- modeling suggests they all came from a depth of at least 50 meters
- many microbes that live in rocks need access to sunlight
- also many microbes are not adapted to live inside rocks
- also most of the martian subsurface is very uninhabitable
- uniform -73°C at a depth of 12 centimeters or deeper
- except at geothermal hot spots if any
[IN NEPA BOTH]

Also the most recent meteorites arriving from Mars today came from the Zunil crater impact somewhere around 700,000 years ago by direct crater count (Do young martian ray craters have ages consistent with the crater count system? : 626) .

This is from rocks from at least 3 meters below the surface (another source says at least 5 meters) by the low levels of radioisotopes produced by cosmic radiation (Ejection ages from krypton‐81‐krypton‐83 dating and pre‐atmospheric sizes of Martian meteorites : 1355). Impact modeling may suggest a depth of 50 to 100 meters below the surface (Ages and geologic histories of Martian meteorites : 152). There’s other confirmatory evidence that they come from at least 1 meter below the surface (The role of target strength on the ejection of martian meteorites : 3) because they don’t show any sign of ionizing radiation from the sky on one side of the rock.

In a scenario with present day life in the surface dirt of Jezero crater, there may be many species that couldn’t get into rocks meters below the surface even if the subsurface is habitable. Microbes that can live inside rocks are called endoliths. Many terrestrial microbes can't live in rocks. Of those that can, many need to live near the surface of the rock with access to sunlight.

We will see that there are many proposed microhabitats native martian life could inhabit that don't require access to geothermal heat.

The best case scenario for planetary protection is a microbe like b. subtilis - which if it exists on Mars may get here on rare occasions (not proven and was a great surprise at the turn of the century when we found it might be possible)

The best case scenario for planetary protection here is for microbes like b. subtilis that may be able to transfer from Mars to Earth (Cockell, 2008, The Interplanetary Exchange of Photosynthesis : 5) (page 5 of the manuscript). But what matters for invasive species are the ones that can’t get here. For instance most photosynthetic life can’t survive impact shock (Cockell, 2008, The Interplanetary Exchange of Photosynthesis). Mileikowsky et al. who first established the potential for microbes to travel from Mars to Earth, say most microorganisms known wouldn’t be able to travel through space:

Whereas this harsh environment sets a definite barrier for most microorganisms known, some have developed survival strategies, by transforming into a dry state, the so-called anhydrobiosis ...,

(Mileikowsky et al., 2000, Natural transfer of viable microbes in space: 1. From Mars to Earth and Earth to Mars : 392).

However we should look at worst case scenarios for planetary protection - analogy of barn swallows which were already in the Americas - it's the starlings that couldn't cross the Atlantic that cause $1 billion of agricultural damage a year

The example of starlings and barn swallows may help. Barn swallows are like b. subtilis which can cross between planets - because it can fly across the Atlantic. But what matters for planetary protection are the worst case scenarios like starlings that can’t get here.

Starling damage reported to the USDA’s Wildlife Services program averages less than $2 million per year, but this is a fraction of all starling damage. Agricultural damage alone is estimated currently at $1 billion per year. Other damage, such as costs for cleaning and maintaining city centers near roosts, veterinary care and loss of production at CAFOs, and public health care, are unknown. A complete inventory of all economic damage likely would show that the starling is the most economically harmful bird species in the United States

(USDA, 2017, European Starling : 16).

Text on graphic: Some microbes may be able to get from Mars to Earth – what matters for invasive species are the ones that can’t.

Barn swallow - can cross Atlantic

Starling - invasive species in the Americas

Didymosphenia geminatum invasive diatom in Great Lakes and New Zealand, can’t even cross oceans

Microbes can be invasive too. One clear example of an invasive terrestrial diatom is the freshwater diatom "Didymo" (Didymosphenia geminatum) which causes many problems in New Zealand (Spaulding et al., 2010. Diatoms as non-native species).

We need to be prepared to find unfamiliar life on Mars with no common ancestor with terrestrial life
- astrobiologists often say this when they design instruments to search for life on Mars [IN NEPA BOTH]
- NASA's own iMost team agrees

Astrobiologists say we need to be prepared to find unfamiliar life on Mars that has no common ancestor with terrestrial life. That includes the iMOST team you assembled to advise you on the science experiments for the samples from Jezero crater.

“We cannot predict with any accuracy life's form and characteristics, whether it would be viable …, or whether it shares a common ancestor with life on Earth.”

(Beaty et al., 2017, iMOST : 88)

Text on graphic: Zunil crater and Jezero crater marked.

The last meteorites to leave Mars for Earth

• left Zunil crater 700,000 years ago (approx)
• came from at least 3 meters below the surface
• probably from at least 50 meters below

In scenarios with present day Martian life:

• most species in Jezero crater probably can't reach Zunil crater (perhaps biofilms in ultracold brines or in micropores in gypsum)
• most terrestrial microbes can't survive sudden ejection at kms / sec or vacuum
• the surface dust, salts and dirt can't physically become a meteorite
• most species in surface layers probably can't get into deep rocks (e.g. photosynthetic)

We don’t know if ANY LIFE EVER GOT FROM MARS TO EARTH

Background map: Google Mars doesn't seem to have an option to share this exact scene - but this is a zoom in on Jezero crater in Google Mars and Zunil crater in Google Mars

In short, this is a widely held belief, that any Martian species have got to Earth already. However, as we see, it's not a valid argument.

Your former planetary protection officers would have been incapable of making this error.

No we don't have evidence Mars is uninhabitable [IN NEPA BOTH]
- you even plan to test Perseverance's samples to see if any lifeforms detected from Mars are still alive

Also it is incorrect to say we have evidence Mars is uninhabitable

Existing credible evidence suggests that conditions on Mars have not been amenable to supporting life as we know it for millions of years (… National Research Council 2022).
(NASA, 2023, Mars Sample Return FINAL PEIS : 1–6)

If we look at NASA's Mars science goals, the second half of Goal 1 is to determine if Mars still supports life (Mars science goals, objectives, investigations, and priorities: 2020 version : 9).

It is also objective 2.3 for your samples you plan to return from Jezero crater:

• Objective 2.3 for Perseverance samples:
  “Modern biosignatures: Assess the possibility that any lifeforms detected are still alive or were recently alive.” (Planning implications related to sterilization-sensitive science investigations associated with Mars Sample Return (MSR) : table 2)

Your scientists plan to

• test these very samples for metabolism and respiration (Beaty et al., 2017, iMOST : 92).
• experiment to see if they can get anything to grow from them (Beaty et al., 2017, iMOST : 93).

Your own cite for “existing credible evidence" that Mars is uninhabitable is actually about searches to see if "habitable regions currently exist " on Mars

Even your own cite for “existing credible evidence” is the opposite of evidence. Your sentence:

Existing credible evidence suggests that conditions on Mars have not been amenable to supporting life as we know it for millions of years (… National Research Council 2022).
(NASA, 2023, Mars Sample Return FINAL PEIS : 1–6)

Your 2022 cite:

“The exploration of … Mars … will help establish whether localised habitable regions currently exist within these seemingly uninhabitable worlds”. (Origins, Worlds, and Life : 393 [Click on the X to go straight to page 393]).

The reader isn’t alerted to these discrepancies.

The biological safety report similarly looks only at best cases for planetary protection
- a systemic issue
- needs someone on the team trained to search carefully for worst case scenarios as they miss several counterexamples that aren't hard to find [NEW]

Before I mention issues with the Biological safety report, I'd like to thank the sterilization working group for the effort formulating their position in a scientifically precise way with example diseases. Some of the counterexamples were often subtle and in some cases they led my survey to topics that seem new to the planetary protection literature. As an example - following up their example of Candidas lead to the counterexample of Aspergillus which I mention below. They also raised the issue of prions for the first time since 1997. See:

• A new size limit review could look at prions - raised by the biosafety report and last considered in 1997 because of the very recent discovery of prions in all three of the domains of life, archaea, bacteria and eukaryotes and so probably in the last common universal ancestor too [NEW]

There are various other points of interest in the report.

The issues I found in the biological safety report were more of a systemic nature. They were rather to do with their overall approach to risk assurance, of looking for best case scenarios and then looking no further once they found them, which showed a lack of training in risk assurance and is a side effect of NASA's decisions to close down its planetary protection office and other sources of peer review that would have spotted the mistakes. .

Though the counterexamples were often subtle and hard to spot, there were several counterexamples in the literature that they could have found relatively easily if anyone had searched for them. It's more an issue with the approach. To add to the remark of your first planetary protection officer, John Rummel:

People have to have some kind of respect for the unknown. If you have that respect, then you can do a credible job, and the public is well-served by your caution.”

(Controversy Grows Over whether Mars Samples Endanger Earth)

I think it's also to do with asking the right question. To serve the public well you need to ask the question:

"Let's see if there is any way this house CAN have a house fire even though the risk seems low".

In worst case scenarios microbes do NOT need to have long-term evolutionary contact with Earth hosts to harm us - example of tetanus which kills thousands of unvaccinated newborns every year and legionnaires disease which is a disease of protozoa and biofilms not adapted to invade lungs which also kills

“Since any putative Martian microorganism would not have experienced long-term evolutionary contact with humans (or other Earth host), the presence of a direct pathogen on Mars is likely to have a near-zero probability.”

(Craven et al., 2021, Biological safety : 6)

Your biological safety group focuses on best case scenarios for planetary protection of diseases adapted to humans, even considering diseases like yellow fever transmitted to humans from monkeys via mosquitoes (Craven et al., 2021, Biological safety : 6).

The report shows no awareness of counter examples in the literature like tetanus, which kills thousands of unvaccinated newborns a year (Tetanus) and is not adapted to infect any organism, and Legionnaires disease, only adapted to infect protozoa and also able to live outside protozoa in a biofilm, and many others in the report by Warmflash et al. (Warmflash et al, 2007, Assessing the Biohazard Potential of Putative Martian Organisms for Exploration Class Human Space Missions, 14–15)

Warmflash et al. were discussing the potential for astronauts on Mars to be harmed by extant microbes there. They concluded that we should accept these risks because in their view they are outweighed by the benefits of human exploration of Mars. They suggested we contain them as far as possible, through biological .containment on Mars and quarantine on return to Earth., writing:

Since the discovery and study of Martian life can have long-term benefits for humanity, the risk that Martian life might include pathogens should not be an obstacle to human exploration.

(Warmflash et al, 2007, Assessing the Biohazard Potential of Putative Martian Organisms for Exploration Class Human Space Missions, 2)

However the Biological Safety Report sidesteps the whole discussion - the report shows no awareness that there is any possibility of harm of this type.

For more on this see below: Invalid use of ten examples of pathogens that co-evolved with humans or other Earth hosts to conclude that Mars is likely to have near zero probability of a direct pathogen - there are many counterexamples of pathogens that didn't co-evolve with humans or any other Earth host like tetanus or Aspergillus fumigatus

My suggestion in this open letter is that with modern technology we no longer need to balance the value of human colonization of Mars or a Mars sample return against low risks of possibly widespread harm to human health.

In worst case scenarios microbes that live in extreme conditions on Mars would be pre-adapted to warm conditions on Earth - the Biological Safety Report's own cite has an example of a microbe from the Canadian permafrost isolated at -16°C, may be able to grow below -25°C, with optimal growth at 25°C and able to grow up to human blood temperature

The biological safety report focuses on best case scenarios for planetary protection of microbes that live only in extreme conditions on Earth reasoning:

“There are many described extremophiles that may survive in environments that are extreme to human or animal life (e.g. extremes of temperature or pressure) but do not survive under conditions in our normal habitat (Merino et al. 2019) … Thus, it is plausible that any Martian microbe, after it arrives on Earth, would not be viable on Earth due to a lack of its required Martian nutritional and environmental conditions.”

(Craven et al., 2021, Biological safety : 6)

This shows no awareness of many examples of terrestrial microbes that do well in Mars simulation chambers, which suggests the same is possible in reverse

Their only cite, Merino et al, has only one example that's a plausible Mars analogue in temperature range and it is a counterexample: Planococcus Halocryophilus: (Living at the extremes: extremophiles and the limits of life in a planetary context: table 3) Optimal growth at 25°C and up to 37°C. Showed metabolic activity down to -25°C, the lowest temperature tested ((Bacterial growth at− 15°C …) and may grow at ultra low temperatures too slowly to test in the laboratory). isolated from Canadian permafrost with ambient temperature around -16°C . (Merino et. al., 2019, Bacterial growth at− 15 C; molecular insights from the permafrost bacterium Planococcus halocryophilus Or1)

Text on graphic: Merino et al.'s table of extremophiles has one remarkable exception with a very broad temperature range

Optimal growth 25°C. Metabolic activity at least to -25°C. Found at -16°C.

(Merino et al., 2021, Living at the extremes: extremophiles and the limits of life in a planetary context.: table 3)

Jezero crater may be more habitable to Martian life than NASA's cite SR-SAG2 suggests
- NASA and ESA commissioned a Space Studies Board review of it out of a perception in some circles that the authors of SR-SAG2 were too closely aligned with NASA's Mars Program Office
- the review found many knowledge gaps SR-SAG2 didn't address directly relevant to Jezero crater [IN NEPA BOTH]

SR-SAG2 seems to be better known than the Space Studies Board review of it. SR-SAG2 has 308 cites in Google Scholar. The Space Studies Board review of it has only 16 cites in Google scholar.

So it's an understandable omission to leave out this review -- but it is serious omission from an Environmental Impact Statement given that NASA and ESA commissioned the report because of a perception in some circles that the views of the MEPAG group were too closely aligned to the Mars Program office (Space Studies Board, 2015,  Review of the MEPAG report on Mars special regions. : xi – xii). The Space Studies Board found many ways that Mars could be more habitable than suggested in SR-SAG2.

The draft EIS singles out Jezero crater as a place on the Martian surface which they say is especially inhospitable for life to survive

Consensus opinion within the astrobiology scientific community supports a conclusion that the Martian surface is too inhospitable for life to survive there today, particularly at the location and shallow depth (6.4 centimeters [2.5 inches]) being sampled by the Perseverance rover in Jezero Crater, which was chosen as the sampling area because it could have had the right conditions to support life in the ancient past, billions of years ago (Rummel et al. 2014, Grant et al. 2018).

[Rummel et al., 2014 is often referred to as SR-SAG2]

( NASA, 2022 : 1-6):

Their cite Grant et al seems to be a mistake. It’s about the geographic features of the landing site. It briefly mentions in one sentence that Vastitas Borealis was rejected partly for planetary protection reasons because of subsurface ice. That's it. But the other cite Rummel et al is SR-SAG2.

NASA and ESA took the unusual step of commissioning a review of this study partly in response to concerns its authors were too closely aligned with the Mars Program office

NASA and ESA commissioned this review of SR-SAG2 partly out of concerns that MEPAG is not independent from NASA. When they found out that they had similar concerns they commissioned a combined review.

There were two reasons why both agencies took the seemingly unusual step of independently commissioning reviews of a review paper that was to be published in a peer-reviewed journal.

First, there is the perception in some circles that MEPAG is not independent and that its views are too closely aligned with NASA’s Mars Program Office.

(Space Studies Board, 2015, Review of the MEPAG report on Mars special regions. : xi – xii).

NASA responded to my comment on this omission. They don't see the significance of #this review for planetary protection. They see the knowledge gaps identified as minor. They don't consider any of them .

Even to the extent that habitability and special regions are considered together, Jezero Crater’s shallow subsurface has parameters for neither.

(NASA, 2023, MSR FINAL PEIS : B-73)

We will see in the following sections that the Space Studies Board paints a picture of a potentially far more habitable Mars especially in locations such as Jezero crater identifying many knowledge gaps in SR-SAG2, including:(see below)

• that maps made from orbit represent an incomplete state of knowledge about habitability and are subject to change as our understanding of Mars changes
   
• that SR-SAG2 didn't adequately discuss transport in the atmosphere
   
• that SR-SAG2 only briefly considered implications of lack of knowledge of microenvironments - it gave a list of seven potential types of microenvironment that may occur on the surface of Mars but then took this no further
   
• The Space Studies Review of SR-SAG2 draws special attention to biofilms - where many microbes work together to build a microbial "house" by exuding plastic-like substances called EPS to protect themselves - and exude various biochemicals to make their new homes more habitable and protect themselves from adverse conditions much like the way we use our houses

Many of the knowledge gaps identified in the Space Studies Board review of SR-SAG2 are highly significant for Jezero crater

I will go into this in some depth because the SR-SAG2 picture is so widely cited and the Space Studies Board review isn't so well known. It paints a picture of a potentially far more habitable Mars especially when combined with new discoveries since 2015 about potential new microhabitats, biofilms and transport through the atmosphere amongst others.

Indeed,the (Space Studies Board, 2015, Review of the MEPAG report on Mars special regions) modified all the main conclusions from SR-SAG2 relevant to NASA's statement about Jezero crater in the EIS. This is why it was such a serious error to refer to SR-SAG2 without mentioning the Space Studies Board review of it.

This emphasis on SR-SAG2 in the literature may have contributed to a common belief that Mars is highly likely to be uninhabitable amongst many space enthusiasts. You get a very different picture when you read the report of the ( Mars Extant Life: What's Next? Conference Report) ( html), a three day conference held in 2019 to discuss potential for present day life on Mars.

A significant subset of conference attendees concluded that there is a realistic possibility that Mars hosts indigenous microbial life. A powerful theme that permeated the conference is that the key to the search for martian extant life lies in identifying and exploring refugia (“oases”), where conditions are either permanently or episodically significantly more hospitable than average. Based on our existing knowledge of Mars, conference participants highlighted four potential martian refugium (not listed in priority order): Caves, Deep Subsurface, Ices, and Salts.”

They are relying on information that is not widely known, perhaps in part due to the popularity of SR-SAG2.

- that maps made from orbit represent an incomplete state of knowledge about habitability and are subject to change as our understanding of Mars changes

The Review of MEPAG warns that maps such as the ones NASA relied on to select Jezero crater as a landing site represent an incomplete state of knowledge

Maps that illustrate the distribution of specific relevant landforms or other surface features can only represent the current (and incomplete) state of knowledge for a specific time—knowledge that will certainly be subject to change or be updated as new information is obtained

(Space Studies Board, 2015,  Review of the MEPAG report on Mars special regions.  : 28):

- that SR-SAG2 didn't adequately discuss transport in the atmosphere

 The Space Studies Board Review of MEPAG also says SR-SAG2 didn’t adequately discuss the potential for life to be transported in the dust in the atmosphere (e.g. dust storms)

"The SR-SAG2 report does not adequately discuss the transport of material in the martian atmosphere. The issue is especially worthy of consideration because if survival is possible during atmospheric transport, the designation of Special Regions becomes more difficult, or even irrelevant."

(Space Studies Board, 2015,  Review of the MEPAG report on Mars special regions. :12).

 Here, “special regions” are regions where terrestrial organisms are likely to propagate. The second half of the definition isn’t used much given that we don’t yet know capabilities of any putative Martian life:

“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 form
s.”

(Space Studies Board, 2015,  Review of the MEPAG report on Mars special regions. ::6)

If terrestrial life can be spread from anywhere to anywhere on Mars it becomes much harder or impossible to map out safe regions for forward contamination, depending how easily it can spread

- that SR-SAG2 only briefly considered implications of lack of knowledge of microenvironments - it gave a list of seven potential types of microenvironment that may occur on the surface of Mars but then took this no further

The Space Studies Board review of MEPAG says SR-SAG2 only briefly considered the implications of our lack of knowledge of microenvironments on Mars

Physical and chemical conditions in microenvironments can be substantially different from those of larger scales. Although the SR-SAG2 report considered the microenvironment (Finding 3-10), the implications of the lack of knowledge about microscale conditions was only briefly considered.

(Space Studies Board, 2015,  Review of the MEPAG report on Mars special regions. :12).

This is what the 2014 report said (Rummel et al. , 2014:904).

Finding 3-10: Determining the continuity/heterogeneity of microscale conditions over time and space is a major challenge to interpreting when and where Special Regions occur on Mars.

(Rummel et al., 2014, A new analysis of Mars “special regions”: findings of the second MEPAG Special Regions Science Analysis Group (SR-SAG2) : 904)

It then gives a list of seven naturally occurring microenvironments on Mars: Vapor-phase water available Vapor or aerosols in planet’s atmosphere; within soil cavities, porous rocks, etc.; within or beneath spacecraft or spacecraft debris

• Ice-related Liquid or vapor-phase water coming off frost, solid ice, regolith or subsurface ice crystals, glaciers
• Brine-related Liquid water in deliquescing salts, in channels within ice, on the surface of ice, within salt crystals within halite or other types of ‘‘rock salt’’
• Aqueous films on rock or soil grains Liquid water on regolith particles of their components such as clay minerals, on surface of ice, on and within rocks, on surfaces of spacecraft
• Groundwater and thermal springs (macroenvironments) Liquid water
• Places receiving periodic condensation or dew Liquid water on regolith particles of their components such as clay minerals, on surface of ice, on and within rocks, on surfaces of spacecraft
• Water in minerals Liquid water bound to minerals

(Rummel et al., 2014, A new analysis of Mars “special regions”: findings of the second MEPAG Special Regions Science Analysis Group (SR-SAG2) : 904)

The 2015 Space Studies Board review says that though SR-SAG2 considered these microenvironments it only briefly considered the implications of our lack of knowledge of them

Physical and chemical conditions in microenvironments can be substantially different from those of larger scales. Although the SR-SAG2 report considered the microenvironment (Finding 3-10), the implications of the lack of knowledge about microscale conditions was only briefly considered.

…

Craters, and even microenvironments underneath and on the underside of rocks, could potentially provide favorable conditions for the establishment of life on Mars, potentially leading to the recognition of Special Regions where landscape-scale temperature and humidity conditions would not enable it.

…

The review committee agrees with Finding 3-10 of the SR-SAG2 report but stresses the significance of the microenvironment and the role it might play on the definition of a Special Region in areas that (macroscopically speaking) would not be considered as such.

(SSB, 2015, Review of the MEPAG report on Mars special regions : 11 - 12 ).

- example of the brines found by Curiosity

This was a surprise discovery by Curiosity using its Dynamic Albedo of Neutrons instrument. This detected liquid water at night and even through to 6 am on some days with sufficient water for living processes, but too cold for life, on the surface as well as slightly below the surface. (Transient liquid water and water activity at Gale crater on Mars : figure 3a and 3c),

This is an example of the SR-SAG2

“Brine-related Liquid water in deliquescing salts, in channels within ice, on the surface of ice, within salt crystals within halite or other types of ‘rock salt’”

(Rummel et al., 2014, A new analysis of Mars “special regions” : findings of the second MEPAG Special Regions Science Analysis Group (SR-SAG2) : 904)

These have enough water for life through to around 6 am on the surface but are too cold. Later in the same day they are warm enough for life but would be far too dry. The main question is, is there any way that life that has evolved on Mars for billions of years could have found a way to connect together those two conditions on the same day to make a habitat for itself that is both wet enough for life and warm enough. There one possibility is that Martian life is able to exploit water at far lower temperatures than terrestrial life. It's likely that terrestrial life can grow below -20°C very slowly but -70°C is so low that it's unlikely terrestrial life could exploit it even with doubling times of millennia or more. But maybe a different biochemistry could, perhaps using perchlorates internally, a biochemistry suggested by Schulze-Makuch et al. (Schulze-Makuch et al.. 2010. A perchlorate strategy for extreme xerophilic life on Mars).

Another possibility is that biofilms might make them habitable, so I'll discuss this in the section on biofilms below.

• Could there be oases for these biofilms even in Jezero crater? Perhaps Nilton Rennó's suggestion of biofilms exploiting the brines Curiosity found could describe a suitable oasis?

- example of water condensing in micropores in salts found in the Atacama desert that may be relevant to Mars

NASA's second planetary protection officer, Cassie Conley has mentioned another possible habitat in places like Gale crater and Jezero crater:

From the perspective of planetary protection, Conley is also concerned about terrestrial organisms that can absorb water from the air. She recalls fieldwork she did in the Atacama Desert in Chile, which is one of the driest places on Earth, with less than 0.04 inch of rain a year.

Even in this desiccated place, she found life: photosynthetic bacteria that had made a home in tiny chambers within halite salt crystals. There’s a small amount of water retained inside the halite and, at night, it cools down and condenses both on the walls of the chambers and on the surface of the organisms that are sitting there.

(Strauss, 2016, Going to Mars Could Mess Up the Hunt for Alien Life)

These are an example of the SR-SAG2

Vapor-phase water available Vapor or aerosols in planet’s atmosphere; within soil cavities, porous rocks, etc.; within or beneath spacecraft or spacecraft debris

(Rummel et al., 2014, A new analysis of Mars “special regions”: findings of the second MEPAG Special Regions Science Analysis Group (SR-SAG2) : 904)

Jezero crater doesn't have the massive white salt deposits of Gale crater but it has gypsum, in small quantities at least. So it could have life using micropores in gypsum. This increases the humidity of the air significantly and water condenses inside the pores.

There is a fair bit of research now into these micropore habitats, including gypsum and salt. The first paper is from 2012: ( Wierzchos, et al., 2012, Novel water source for endolithic life in the hyperarid core of the Atacama Desert) just one year after the most recent NRC Mars sample return report in 2009. Other papers include (Vitek et al., 2012, Microbial colonization of halite from the hyper-arid Atacama Desert studied by Raman spectroscopy) and Paul Davies wrote an opinion piece about them in the Guardian in (Davies, 2014, The key to life on Mars may well be found in Chile)

This is of especial importance for Martian life as it would have had millions of years to discover and colonize microhabitats such as pores in salt or gypsum, or to adapt to retain water from the brines discovered by curiosity, or to make use of the humidity in the atmosphere.

There are many other new microhabitats suggested for Mars that fit one or other of those seven categories. In some cases the evidence is very strong, or the reasoning is compelling that they should exist though undetectable from orbit.

See: Executive summary highlights (below)

The Space Studies Review of SR-SAG2 draws special attention to biofilms - where many microbes work together to build a microbial "house" by exuding plastic-like substances called EPS to protect themselves - and exude various biochemicals to make their new homes more habitable and protect themselves from adverse conditions much like the way we use our houses

The Space Studies Board review draws special attention to biofilms. These aren’t discussed in SR-SAG2 (it has only one mention of the word).

Given the wide distribution and advantages that communities of organisms have when they live as biofilms enmeshed in copious amounts of EPS [substances that microbes can produce around them to help make a “home” in a hostile environment], it is likely that any microbial stowaways that could survive the trip to Mars would need to develop biofilms to be able to establish themselves in clement microenvironments in Special Regions so that they could grow and replicate.

(SSB, 2015, Review of the MEPAG report on Mars special regions :11)

The 2015 Space Studies Board review starts the biofilm section saying:(SSB, 2015, Review of the MEPAG report on Mars special regions :11).

The SR-SAG2 report identified the ability of microorganisms to withstand multiple stressors as an important area of research.

Here is a graphic to show the idea (not from the SSB board report - they don't have any graphics on biofilms)

Figure 12: Text on graphic: How EPS (extrapolymeric substances) can make a “home” of the hostile Martian surface.

Some of the environment stressors

100% humidity varies to 0%

Heat, cold, UV, dust storms

Oxidants, nutrients

Algae may add oxygen

Retains moisture from night to daytime when temperature soars from -70°C to above 0°C.

Cryoprotectants - protects from cold shock

Extrapolymeric substances (EPS): proteins, DNA, lipids, polysaccharides, other large organic molecules.

A biofilm is like a microbe's "house" which can keep it warm, wet, protected from UV, and which it shares with other microbes

Graphic adapted from figure 2 of (Stream biofilm responses to flow intermittency: from cells to ecosystems) 

Microbes in biofilms can use those organic plastic like substances (EPS) to inhabit ecological niches that would otherwise be uninhabitable (SSB, 2015, Review of the MEPAG report on Mars special regions :11)

The majority of known microbial communities on Earth are able to produce EPS, and the protection provided by this matrix enlarges their physical and chemical limits for metabolic processes and replication. EPS also enhances their tolerance to simultaneously occurring multiple stressors and enables the occupation of otherwise uninhabitable ecological niches in the microscale and macroscale.

Illustrative scenario suggested by Mosca et al combining three of these ideas and relevant to Jezero crater - biofilms that formed in the past on Mars when it was more habitable and propagated ever since as fragments blown in the winds on Mars perhaps only succeeding occasionally in establishing a foothold in a new microenvironment

Combining these ideas we get an especially interesting scenario for Jezero crater which combines together three of those knowledge gaps, transport in the atmosphere, biofilms and microhabitats. Amongst several relevant discoveries, later research found small fragments of biofilm, thin layers of a microbial colony three hundredths of a millimeter thick, can travel 100 kilometers in daylight in the light Martian winds before it is sterilized (Billi et al.., 2019, A desert cyanobacterium under simulated Mars-like conditions in low Earth orbit: implications for the habitability of Mars)

(Asma et al., 2022, (An Overview of Biofilm Formation–Combating Strategies and Mechanisms of Action of Antibiofilm Agents : Figure 1)

Mosca et al. suggest that a biofilm could still propagate on Mars in this way as complete biofilm fragments, even if local conditions don’t permit it to establish a biofilm today by slowly growing from a few microbes. All that is needed is that at some time in the past biofilms were able to form, propagating ever since then using these broken off fragments (Mosca et all, , 2019, (Over-expression of UV-damage DNA repair genes and ribonucleic acid persistence contribute to the resilience of dried biofilms of the desert cyanobacetrium Chroococcidiopsis exposed to Mars-like UV flux and long-term desiccation)

These authors don't look at transport at night (the winds are as strong at night as in daytime during Martian dust storms) or in the dust storms which blocked out 97% of the UV for three weeks in the great dust storm of 2018. These could turn the survivable distance from 100 km to 1000s of kilometers.

See: Executive summary highlights (below)

Could there be oases for these biofilms even in Jezero crater? Perhaps Nilton Rennó's suggestion of biofilms exploiting the brines Curiosity found could describe a suitable oasis?

This biofilm scenario is already plausible for somewhere on Mars, so long as there are microhabitats that life can exploit as oases (refugia) on the surface. If these biofilms are spreading in the wind they may sometimes reach Jezero crater from far away on Mars.

However it could also be a plausible scenario even for Jezero crater if we can find a plausible oasis for the biofilms there. We have had many surprises (e.g. the Phoenix leg droplets (Rennó et al., 2009, Possible physical and thermodynamical evidence for liquid water at the Phoenix landing site), so

• it's possible we find a new type of oasis that nobody has thought of.
• It's also possible Martian life found a way to survive on Mars that nobody has thought of yet.

But we can sketch out one plausible type of oasis that we may find in places in Jezero crater based on what we know already.

One idea is Nilton Rennó's suggestion that a biofilm could make the brines Curiosity found habitable. This was another surprise discovery on Mars. Curiosity found liquid water in the salts that take up water at night - on the surface through to 6 am on the same day that it measured surface brines for the last time in the year, it registered a midday temperature of 15 °C (Transient liquid water and water activity at Gale crater on Mars : figure 3a and 3c) Those brines are habitable but too cold for terrestrial life at -73°C at 6 am on that day. But could life somehow retain that water through to warmer conditions?

These brines are an example of the SR-SAG2

“Brine-related Liquid water in deliquescing salts, in channels within ice, on the surface of ice, within salt crystals within halite or other types of ‘rock salt’”

(Rummel et al., 2014, A new analysis of Mars “special regions”: findings of the second MEPAG Special Regions Science Analysis Group (SR-SAG2) : 904)

Nilton Rennó is an expert on Mars surface conditions, who was part of the team that discovered the Phoenix lander drops, principle investigator for Phoenix and who also runs the REMS weather station on Mars for Curiosity. He is co-author of a major review of the current evidence for water and brines on Mars ((Water and brines on Mars: current evidence and implications for MSL)

Nilton Rennó i suggested in an interview that microbes might use biofilms to inhabit these brines

"Life as we know it needs liquid water to survive. While the new study interprets Curiosity's results to show that microorganisms from Earth would not be able to survive and replicate in the subsurface of Mars, Rennó sees the findings as inconclusive. He points to biofilms—colonies of tiny organisms that can make their own microenvironment.

(“Mars liquid water: Curiosity confirms favorable conditions”)

Perseverance can't detect these brines as it doesn't have Curiosity's Dynamic Albedo of Neutrons (DAN) instrument (DAN for Scientists), but calculations suggest they would also be found in Jezero crater; and last longer before drying out than they do in Gale crater (Global Temporal and Geographic Stability of Brines on Present-day Mars : Figure 7). Also as with Gale Crater, the ground temperature in Jezero crater often varies from well below -70 °C to well above 15 °C in a single day as measured by Perseverance (Diurnal variation of the surface temperature of Mars with the Emirates Mars Mission: A comparison with Curiosity and Perseverance rover measurements : Figure 3).

As with microenvironments in terrestrial deserts - most likely only some of the brines would be inhabited. Even though this salty water can be found almost every where there would be Microbial oasis amongst them

• variation in composition of salts,
• variation in local conditions such as fissures in the rock, microcaves, and
• micropores in salt deposits

This would lead to more habitable patches of the brines. In Mars analogue deserts sometimes life is very patchy - it could be even more patchy on Mars with microbial oases kilometers or tens of kilometers apart or more - yet that could be enough for an occasional viable spore to reach Perseverance's sample, perhaps just a few spores in the entire collection.

In the oases where biofilms set up home on Mars, these would likely be a mix of many species working together for extra resilience like the grit crust in the Atacama desert (The grit crust: A poly-extremotolerant microbial community from the Atacama Desert as a model for astrobiology)

In the preprint I try to flesh out one way this could happen by combining this with recent research that finds that a moss Grimmia sessitana collected in the alps did well in a terrestrial Mars simulation chamber in 2019 (Mosses in low Earth orbit: implications for the limits of life and the habitability of Mars) and many other tests for a potential Mars analogue organisms.

Some desert mosses are especially good at absorbing water quickly and then retaining it so that it doesn't evaporate again. Could Mars have moss like organisms that absorb water fast in the early morning when Curiosity found ultra cold salty water even on the surface at -73°C - and retain it even through to midday on the same day when temperatures reached over 15°C?

The desert moss Syntrichia caninervis which is found in desert biocrusts throughout the world uses microgrooves rather than micropores, with an “upside-down” water collection system that collects water droplets which condense onto microgrooves within its leaf hair points and it rapidly funnels those down to the plant below (Effects of leaf hair points of a desert moss on water retention and dew formation: implications for desiccation tolerance). In this way it can absorb water in seconds and retain it for a long time.

Video: Demystifying desert moss hydration

The leaf hairs

• absorb water through capillary action which would otherwise evaporate, which slows down evaporation
• make the mosses more reflective, reflecting away sunlight
• expand as they hydrate and so form a 3D mesh which blocks evaporation
  (Effects of leaf hair points of a desert moss on water retention and dew formation: implications for desiccation tolerance)

This reduces evaporation from the hydrated moss for as long as it has high water content.

Its microgrooves are like a biological analogue of the Atacama salt and gypsum pillars micropores. So - might not Martian life have developed similar structures over billions of years of evolution on Mars? Microgrooves or micropores, or some other structures optimized to collect and retain water in cold conditions, passively, mechanically when it is too cold for metabolic processes.

If there is some biofilm or organism that exploits the brines Curiosity found, it need not closely resemble terrestrial desert mosses. However these mosses could turn out to be our closest terrestrial analogue.

I elaborate on this some more with some other thoughts along with some other ideas, see Executive summary highlights (below).

A common misconception about perchlorates - some microbes are harmed by perchlorates but for others they make Mars more habitable
- a giant dinner plate in the words of Cassie Conley, NASA's second planetary protection officer [IN NEPA BOTH]

This is a common misconception about Mars - that the perchlorates make it uninhabitable. It's the opposite. For some microbes yes, they make it less habitable but for others they make Mars MORE habitable.

Cassie Conley put it like this in 2015.

The salts known as perchlorates that lower the freezing temperature of water at the R.S.Ls, keeping it liquid, can be consumed by some Earth microbes. “The environment on Mars potentially is basically one giant dinner plate for Earth organisms,” Dr. Conley said.
(Mars Is Pretty Clean. Her Job at NASA Is to Keep It That Way)

There are many reports of perchlorate reducing microbes from perchlorate contaminated soils. However they also occur naturally in some places that are naturally enriched in perchlorates (Evidence for Biotic Perchlorate Reduction in Naturally Perchlorate-Rich Sediments of Pilot Valley Basin, Utah)

If there is life on Mars, either the native Martian life actually uses it, or it is very resistant to oxidants which it has to be anyway with perchlorates and a very oxidising surface. Interestingly the microbe from the Canadian permafrost that the Biological Safety Report missed is actually able to grow in 10% by weight perchlorate solution.

Furthermore, our results show the development of cohesive biofilms in perchlorate-rich media (Fig. 4A)

(Bacterial Growth in Chloride and Perchlorate Brines: Halotolerances and Salt Stress Responses of Planococcus halocryophilus)

See also

• Yes perchlorates are bacteriocidal for b. subtilis when irradiated with UV - but the effects are far less at lower temperatures or when mixed with dirt or dust - a shadow under a rock or a few microns of dirt eliminates most of the UV as do dust storms - and the organic plastic compounds exuded by biofilms (EPS) and other modifiers would protect microbes against oxidants like chlorates, and chlorites, or indeed hydrogen peroxide

The 3-day conference in 2019 "Extant Life on Mars - What's Next?" mentions only one sample return of special interest as a habitat for present day life - surface salts including perchlorates

The report of the 3 day conference on the potential for present day life on Mars, held in Carbland New Mexico on November 5-8 mentions only one specific suggestion for a Mars sample return to look for present day life. It suggests that salts are a prime target both for their preservation potential for recent life and as a habitat for present day life. It specifically mentions perchlorates as a potential habitat.

If life exists today on Mars, or existed in the recent past, microscopic examination of such evaporite minerals and their fluid inclusions might offer a straightforward and realistic way to confirm its existence, while spectroscopic analyses may uncover biochemicals that represent biosynthetic or breakdown products of life.

Moreover, having these features as potential targets for future Mars Sample Return efforts would allow for potentially concentrated organics and extant life analyses within its mineral structure.

Salts and brines containing concentrated dissolved solutes and gas pockets could serve as energy and nutrient resources for life (e.g., perchlorates, nitrates, sulfates, organics, methane), with near-surface evaporites additionally providing access to sunlight for potential phototrophic activities.

Consequently, when considering NASA’s current mission concepts, evaporitic environments at the surface and near subsurface offer targets that are likely to be readily accessible to robotic exploration.

( Mars Extant Life: What's Next? Conference Report : 797) ( html)

These four plausible but invalid arguments reinforce false beliefs in the space community - which they use in good faith to recommend dropping all planetary protection for Mars and Earth [IN NEPA BOTH]

[I cover this in the first round of comments in the attachment in a section about Zubrin's arguments]

These arguments reinforce false beliefs in the space community. Many find them plausible. They are already used, in good faith but incorrectly, to recommend dropping all planetary protection for samples from Mars, for instance by Robert Zubrin, president of the Mars society (Zubrin, 2000, Contamination From Mars: No Threat) with the response from planetary protection experts (Rummel et al., 2000, Opinion: No Threat? No Way : 4 - 7). So - though they likely don't realize what they are doing, this EIS is effectively endorsing Robert Zubrin's invalid arguments, and supporting him in opposition to their own first planetary protection officer John Rummel and other experts on planetary protection. John Rummel probably has more published on a Mars sample return than any other living author - and Robert Zubrin only has that non peer reviewed op. ed.

These are Zubrin's four plausible but invalid reasons for dropping all planetary protection. They are affirmed and not countered in this EIS: I summarize briefly why they are in valid in square brackets, briefly summarized the detailed explanations presented in the previous sections.

• (PLAUSIBLE BUT INVALID) existing credible evidence says Mars has been uninhabitable for millions of years
  [iMOST think there is enough of a chance of finding life to try to get something to grow from the samples  (iMOST : 94) and contradicted by NASA's own cite: "The exploration of … Mars … will help establish whether localised habitable regions currently exist within these seemingly uninhabitable worlds”. (Origins, Worlds, and Life : 393). ]
  
  See above: No we don't have evidence Mars is uninhabitable - you even plan to test Perseverance's samples to see if any lifeforms detected from Mars are still alive and Your own cite for “existing credible evidence" that Mars is uninhabitable is actually about searches to see if "habitable regions currently exist " on Mars
   
• (PLAUSIBLE BUT INVALID) If there is life on Mars it must have got here already (NASA, 2023, MSR FINAL PEIS 3–3),
  [most terrestrial microbes couldn’t get from Mars to Earth and the dirt, dust, salts can’t get here at all, rebutted by NASA's own cite (Planetary protection classification of sample return missions from the Martian moons : 45) ]
  
  See above: Microbes from Jezero crater do NOT get to Earth better protected and faster in Mars meteorites
    

• (PLAUSIBLE BUT INVALID) If there is life on Mars, it is harmless to humans (Biological safety : 6)
  [some microbes that harm humans are not adapted to infect any organism, example tetanus which kills thousands of unvaccinated newborns a year (Tetanus)  (Warmflash et al, 2007, Assessing the Biohazard Potential of Putative Martian Organisms for Exploration Class Human Space Missions, 14–15) ]
  
  See below: Invalid use of ten examples of pathogens that co-evolved with humans or other Earth hosts to conclude that Mars is likely to have near zero probability of a direct pathogen - there are many counterexamples of pathogens that didn't co-evolve with humans or any other Earth host, like tetanus or Aspergillus fumigatus
   
• (PLAUSIBLE BUT INVALID) Anyway, if there is life on Mars, it can’t survive on Earth (Biological safety : 6)
  [Their own cite Merino et al. lists a microbe Planococcus Halocryophilus found in Canadian permafrost at -16 °C, metabolizes at least down to -25°C, has optimal growth at 25°C and can grow up to human blood temperature (Merino et al., 2019, Living at the extremes: extremophiles and the limits of life in a planetary context : table 3)]
  
  See below: In worst case scenarios microbes that live in extreme conditions on Mars would be pre-adapted to warm conditions on Earth - the Biological Safety Report's own cite has an example of a microbe from the Canadian permafrost isolated at -16°C, may be able to grow below -25°C, with optimal growth at 25°C and able to grow up to human blood temperature

It is possible to describe a consistent set of beliefs to explain this draft EIS, based on these four invalid arguments, and especially the Mars meteorite, not spotting that their cite for this argument rebutted it, and a knowledge of SR-SAG2 and not the much less well known Space Studies Board review of it, which is not cited in the EIS.

1. (BASED ON SR-SAG2 - NOT AWARE OF KNOWLEDGE GAPS IN SPACE STUDIES BOARD REVIEW AND RECENT RESEARCH) we almost certainly find no life on Mars.
   (If so, the "existing credible evidence" is hyperbole - the authors surely know the second part of NASA's first objective is to search for present day life on Mars but they simplify this for the public to no chance instead of low chance).
    
2. (BASED ON INVALID METEORITE ARGUMENT) if we do find life there, it will just be like life from a terrestrial desert (based on a false belief in a scientific consensus that any Martian life already got here on a Mars meteorite)
    
3. (BASED ON INVALID METEORITE ARGUMENT) Or there may be a chance we find alien life or novel life on Mars, if so then it has already got here on meteorites so we know in advance that it couldn't survive on Earth.

If one believed those things then the EIS would make sense. But it relies heavily on the Mars meteorite argument which of course is invalid.

I.e. any Mars life they think is like terrestrial life but adapted to an environment they think doesn't exist on Earth or it's already here.

At least that is the picture one gets from reading the draft EIS.

The most important knowledge gaps in the EIS then are:

• Jezero crater may be more habitable to Martian life than NASA's cite SR-SAG2 suggests - NASA and ESA commissioned a Space Studies Board review of it out of a perception in some circles that the authors of SR-SAG2 were too closely aligned with NASA's Mars Program Office - the review found many knowledge gaps SR-SAG2 didn't address directly relevant to Jezero crater
   
• Microbes from Jezero crater do NOT get to Earth better protected and faster in Mars meteorites

t's for NASA to find out what went wrong and how the EIS got so many mistakes in it. But this is one understandable way that it could have happened so this is to help others to understand how it's possible - just a result of not having the necessary experts on the team and knowledge gaps and a lack of familiarity of the planetary protection literature.

These are extremely serious mistakes because of the high regard NASA enjoys for planetary protection due to its previous excellence in the field - if this EIS is not withdrawn by NASA, other actors in good faith but mistakenly could use just one of those arguments to drop all planetary protection both ways for missions to Mars

I can’t overstress how extremely serious these mistakes are, because of the precedent and respect for NASA and its previous reputation in the field of planetary protection. Everyone looks to NASA for the lead on planetary protection.

If just one of those arguments was indeed valid, we could drop all planetary protection of Earth. This EIS is a potential precedent for other actors, in good faith, to return samples from anywhere on Mars with no protection of Earth’s biosphere.

These serious mistakes also lead the EIS to the judgement "that the potential environmental impacts would not be significant" [IN 2ND NEPA]
- and that risks from lab escapes are "negligible" [NEW]
- flying against the consensus of ALL the major studies to date

Not surprisingly after many invalid arguments that many in the space community find plausible, your EIS says:

The relatively low probability of an inadvertent reentry combined with the assessment that samples are unlikely to pose a risk of significant ecological impact or other significant harmful effects support the judgement that the potential environmental impacts would not be significant.

(NASA, 2023, MSR FINAL PEIS :3-16)

It also relies on submitted final Environmental Impact Statements for other biosafety level 4 laboratories. Here the sentence An alternative release path resulting from the contamination of workers leading to direct contact with others (members of the public) was also analyzed! refers to analyses done in previous biosafety level environmental impact statement. NASA's team don't provide any of their own. NASA's team concludes:

While not completely analogous, the results of previous NEPA analyses for BSL-4 facilities have concluded that the hazards associated with the operation of BSL-4 facilities are expected to be minimal.

[These next two sentences refer to the analyses by the National Emerging Infectious Diseases Laboratories, and the . National Bio and Agro-Defense Facility for their approved BSL-4 EIS's - NASA hasn't shared any separate biosafety lab analysis for samples returned from Mars with the draft EIS]

Analyses performed in support of recent NEPA documents [by other agencies for previous BSL-4s] conclude that the risk from accidental release of material from a BSL-4 even under accident conditions that include the failure of protective boundaries (e.g., reduced effectiveness of ventilation filtration systems) are minute and can be described as zero (NIH/DHHS 2005).

An alternative release path resulting from the contamination of workers leading to direct contact with others (members of the public) was also analyzed . Qualitative risk assessments for this mode of transmission [for the two previous EIS's for ordinary BSL-4 labs] have shown that the risk to the public is negligible (NIH/DHHS 2005, DHS 2008).

Should the Proposed Action be chosen, Tier II NEPA analyses of the proposed SRF11 would include analysis similar to those performed for existing BSL-4 facilities.

(NASA, 2023, MSR FINAL PEIS :3-14)

Cites:
NIH/DHHS. (2005). Final Environmental Impact Statement National Emerging Infectious Diseases Laboratories, Boston, Massachusetts. Bethesda, MD: National Institutes of Health, U.S. Department of Health and Human Services.
DHS. (2008). National Bio and Agro-Defense Facility Final Environmental Impact Statement. Washington D.C.: U.S. Department of Homeland Security

[My comments in red]

• The draft EIS has nothing about quarantine
  - based on NEPA statements for other BSL-4s, it says the risk to the public from a typical BSL-4 can be described as zero for direct release and is negligible even if workers get contaminated
  - it has no mention of quarantine or precautions for lab leaks
  - no suggestion that there is anything different about a Mars sample receiving facility and a normal BSL-4
  - the big difference is that in a sample receiving facility we don't know what needs to be contained, what its capabilities are, or even if it is based on terrestrial biology while
  - with a BSL-4 the experimenters know what they are studying

After all since you believe all life on Mars has already got here on meteorites you are understandably so confident that it can't have any exotic life like mirror life you don't mention this possibility, and that any diseases in the samples are pathogens we already have on Earth. But this is not established.

• The final Environmental Impact Statement covers the NEPA requirement to consider all major points of view on environmental effects with a statement that the National Academy of Sciences Mars sample return study in 2009 and Martian moons sample return study in 2019 "affirmed the consensus" that the Mars meteorite argument is valid
  - BOTH STUDIES said clearly this argument is INVALID
  - no mention of Cassie Conley's statement in an official NASA video that the samples will be treated as the most hazardous Earth organisms known
  - no mention of the European Space Foundation recommendation to treat the samples as risk group 4 organisms

ALL major studies to date through to the European Space Foundation study in 2012 that say the potential for even large-scale effects is NOT demonstrably zero
- experts do believe the risk of large-scale harm to human health or the environment is low
- but even a low risk of this nature is HIGHLY SIGNIFICANT

This runs through the planetary protection literature from the beginnings of the discipline, in the late 1950s to 60s, with Joshua Lederberg, Carl Sagan and others, onwards (Meltzer, 2012, When Biospheres Collide : 35, 420). The most recent European Space Foundation (ESF) Mars sample return study in 2012 concurs with the 2009 Mars sample review and indeed all major sample return studies to date:

The Study Group also concurs with another conclusion from the NRC reports (1997, 2009) that the potential for large-scale effects on the Earth’s biosphere by a returned Mars life form appears to be low, but is not demonstrably zero.

(Mars Sample Return backward contamination–Strategic advice and requirements : 20)

In more detail they say:

… it is not possible to estimate a probability that the sample could be harmful or harmless in the classical frequency definition of probability.

However it is possible to establish the risk as low, as a consensus of the beliefs of the experts in the field as represented by their experience.

(Mars Sample Return backward contamination–Strategic advice and requirements : 24)

The 2009 Space Studies Board Mars Sample Return study said they couldn't rule out the possibility that Earth had mass extinctions in the past caused by the delivery of Martian organisms on meteorites
- because there are many past mass extinctions not fully understood
- - they conclude that though the risk appears to be low they can't rule out the possibility of future large-scale effects [such as mass extinctions] caused by life introduced from Mars

This is what the Space Studies Board said in 20009 about the meteorite argument and large-scale effects - that they can't rule it out for the past or for the future based on current evidence. The risk is believed to be low but can't be assessed. It may help to present the main points as bullet points first.

• There must have been transfers of material from Mars to Earth and Earth to Mars numerous times in the past
• So it is possible that terrestrial life was delivered from Earth to Mars, or that life had an independent origin on Mars and then was transferred to Earth
• These transfers must have been especially common in the past [this is during the "late heavy bombardment" after the formation of the Moon with many huge impacts on both planets]
• It is not possible to determine if any viable life got from Mars to Earth.
• There is no evidence in the modern era of large scale or negative effects [on Earth's biosphere / ecosystems] caused by Mars meteorites.
• But we can't discount the possibility that such effects happened in the distant past
• It is not known if putative martian organisms would be able to survive ejection, transit and impact delivery to Earth
• In a scenario with life on Mars it is possible that martian life could be sterilized by shock pressure heating during ejection, or by radiation damage during transit
• Based on current evidence it's not possible to assess past or future negative impacts from the delivery of putative extraterrestrial life.

The Space Studies Board said:

Martian Meteorites, Large-Scale Effects, and Planetary Protection

Impact-mediated transfers of terrestrial materials from Earth to Mars, although considerably less probable than such transfers from Mars to Earth, should also have occurred numerous times over the history of the two planets. Thus, it is possible that viable terrestrial organisms were delivered to Mars at some time during the early history of the two planets. As noted above, it is also possible that if life had an independent origin on Mars, living martian organisms may have been delivered to Earth. Although such exchanges are less common today, they would have been particularly common during the early history of the solar system when impact rates were much higher.

Despite suggestions to the contrary,it is simply not possible, on the basis of current knowledge, to deter- mine whether viable martian life forms have already been delivered to Earth. Certainly in the modern era, there is no evidence for large-scale or other negative effects that are attributable to the frequent deliveries to Earth of essentially unaltered martian rocks. However, the possibility that such effects occurred in the distant past cannot be discounted. Thus, it is not appropriate to argue that the existence of martian meteorites on Earth negates the need to treat as potentially hazardous any samples returned from Mars via robotic spacecraft. A prudent planetary protection policy must assume that a potential biological hazard exists from Mars sample return and that every precaution should be taken to ensure the complete isolation of any deliberately returned samples, until it can be determined that no hazard exists

...

Although exchanges of essentially unaltered crustal materials have occurred routinely through- out the history of Earth and Mars, it is not known whether a putative martian microorganism could survive ejection, transit, and impact delivery to Earth or would be sterilized by shock pressure heating during ejection, or by radiation damage accumulated during transit. Likewise, it is not possible to assess past or future negative impacts caused by the delivery of putative extraterrestrial life, based on present evidence.
(SSB, 2009, Assessment of planetary protection requirements for Mars sample return missions : 47)

The Space Studies Board give no particular example of a way that a novel species from another planet could cause large scale harm
- however the Great Oxygenation Event is a good illustration of how a single microbe transferred between planets could cause major changes to another planet's biosphere and
- It's not known if the Great Oxygenation Event caused a mass extinction or not
- but it is an example to show that a single species can in principle cause major changes to a biosphere as with our scenario of mirror life
- with potential to cause mass extinctions as a result

To make this illustration more concrete, let's use the microbe Chroococcidiopsis. It may be partially responsible for the oxygenation of our atmosphere. One minority view explains the unusual ionizing radiation resistance of Chroococcidiopsis as a natural adaptation of Martian organisms (Pavlov et al., 2006. Was Earth ever infected by Martian biota? Clues from radioresistant bacteria. ) .

This is weak evidence since the ionizing radiation resistance of chroococcidiopsis could be a byproduct of the repair mechanisms that chroococcidiopsis uses for UV resistance and desiccation resistance. Cyanobacteria originated in the Precambrian era. It could have developed these mechanisms back then, when, with no oxygen in the atmosphere, there was no ozone layer to shield out UV radiation (Casero et al., 2020,. Response of endolithic Chroococcidiopsis strains from the poly extreme Atacama Desert to light radiation) (Raman et al., 2006, Analysis of proteins involved in the production of MAA ׳ s in two Cyanobacteria Synechocystis PCC 6803 and Anabaena cylindrica)

However, the early Martian atmosphere was rich in oxygen (Lanza et al, 2016, Oxidation of manganese in an ancient aquifer, Kimberley formation, Gale crater, Mars ) be fore Earth and though much of that may well be due to ionizing radiation from solar storms splitting the water it’s not impossible that it had photosynthetic life.as well.

Benner and others have argued that Mars is a likely origin for life because (Shilling, 2015, Are We Martians After All?,) (Benner, 2015,. The case for a Martian origin for Earth life. ) (Ranjan, 2017, The UV Environment for Prebiotic Chemistry: Connecting Origin-of-Life Scenarios to Planetary Environments : 193))

• it had a larger area of dry land for the wet / dry cycles that may have been important for concentrating organics for early life
• dry conditions are needed for RNA which can’t form in water (water is corrosive for RNA).
• it had boron, and phosphates important for likely early life processes,
• boron combines with the oxygen in carbohydrates to prevent formation of tars

It had molybdates which catalyses reactions that can convert the boron stabilized carbohydrates into ribose, needed for the formation of RNA and early life precursors

The curious thing about the Great Oxygenation Event is how long it took. Maybe it takes 2 billion years to evolve the capability. But there is evidence even the earliest bacteria had started on the path to evolution of Photosystemr II. So it could have evolved far faster on other planets. If so, could it have evolved first on Mars, by chance, and then got here after that? (Oliver et al., 2021, Time-resolved comparative molecular evolution of oxygenic photosynthesis), press release (Dunning, 2021, (Photosynthesis could be as old as life itself)

Whatever the cause of the GOE (most would say late evolution of photosynthesis on Earth), it's an example to show the potential for a single species to transform the biosphere of a planet. See:

It would be a major challenge for photosynthetic life to get from Mars to Earth as Charles Cockell showed. The biggest challenge is that the preferred habitat of photosynthetic life is just below the surface or on the surface of rocks where it can access light. In an experiment he did with chroococcidiopsis attached to the aeroshell of a rocket during re-entry of Earth's atmosphere, he found that none of it survived. Not even the organics survived.

However, he didn't rule out the possibility that it could happen on very rare occasions. He concluded

Thus, the planetary exchange of photosynthesis might not be impossible, but quite specific physical situations and/or evolutionary innovations are required to create conditions where a photosynthetic organism happens to be buried deep within a rock during ejection to survive atmospheric transit.
(Cockell, 2008. (The Interplanetary Exchange of Photosynthesis : Survival of the Filters)

So was this an extinction event? The Great Oxygenation Event might have forced rapid evolution rather than extinction. Early anaerobes may have retreated to anaerobic habitats as obligate anaerobes, which we still have today (Lane, 2015,. The vital question: energy, evolution, and the origins of complex life., page 49. )

However, there is some evidence suggesting extinctions. There is evidence of exceptionally large sulfur reducing bacteria from this time, 20 to 265 µm in size, which also occasionally occur in short chains of cells. This may be part of a diverse ecosystem that predated the GOE ( Czaja et al., 2016. Sulfur-oxidizing bacteria prior to the Great Oxidation Event from the 2.52 Ga Gamohaan Formation of South Africa),. See also Czaja interviewed for University of Cincinnati by Melanie Schefft,

“And this discovery is helping us reveal a diversity of life and ecosystems that existed just prior to the Great Oxidation Event, a time of major atmospheric evolution.”

((Schefft, 2016, Life before oxygen, )

If such an ecosystem existed, most traces of it are gone now. However it seems not impossible that the GOE had major impacts on a prior diverse ecosystem.

There are many other confirmed mass extinctions in the fossil record. In many cases the cause is not fully known or debated leaving it at least hypothetically possible that microbial transfer from Mars could be part of the explanation.

Whether or not this ever happened in the past, this worked example of the Great Oxygenation Event shows how in the worst case scenario, independently evolved life from another planet could lead to large scale transformations of the chemistry of Earth’s atmosphere or oceans, climate and ecosystems. Humans with modern technology would surely survive a gradual transformation of our atmosphere and oceans by a microbe with novel capabilities no terrestrial life has yet explored - but it could make the planet significantly less habitable in the short term for humans and other species.

I shared a concrete example of this possibility for a single species of microbe based on alien biology to transform our biosphere with the mirror life scenario above, in this case gradually changing our normal organics to mirror organics, leading either to a situation with nearly all organics in their mirror form or an equal quantity of mirror and normal organics depending on whether and how the terrestrial life is able to evolve to metabolize mirror life - or possibly the other way round that terrestrial life wins.

It seems a biologically possible scenario that a mirror microbe from a planet resembling Mars somewhere in our universe could transform a biosphere resembling Earth's to a mirror biosphere depending on its capabilities. It's hard to estimate the timescale as it depends on its capabilities and how rapidly it radiates to new ecosystems.

• Detailed illustrative mirror life scenario - an analogue of the blue-green algae chroococcidiopsis
  - one of our best candidates for a Mars analogue organism
  - can find all its nutritional requirements in basalt or gabbro, also sea water, fresh water and soil with strains found almost everywhere on Earth
  - and a mirror life analogue on Mars would likely be adapted to metabolize normal organics because of infall from space

2012 ESF study says Mars samples should be treated as risk group 4 Earth organisms with high individual and community risk, usually no treatment available, until we know more
- and in an official NASA video your former planetary protection officer Cassie Conley says we will treat them as the most hazardous organisms known

The 2012 ESF study goes on to say Mars samples should be treated like risk group 4 organisms (high individual and community risk, highly infectious, no treatment available) until we know more. I.e. we should treat them as significant

While, based on assumptions, some aspects of the release of unsterilised Mars material can be framed in some way, with such a level of uncertainty, unknown (and therefore unexpected) consequences driven by unknown mechanisms are conceivable and by definition are hardly manageable and predictable.

In this context, confinement of the sample appears to be the best prevention method. This principle is also applied when an unknown pathogen with a high case fatality rate is isolated: it is assimilated to Risk Group 4 and contained in laboratories with the highest level of confinement until further knowledge about the pathogen allows it to be down graded to a lower risk group.

Following the same principle, a priori assignment of a Mars sample to Risk Group 4 appears to be the best measure.

.(Amman et al., 2012, Mars Sample Return backward contamination–Strategic advice and requirements : 24)

They summarize Risk Group 4 as:

Risk Group 4 (high individual and community risk): A pathogen that usually causes serious human or animal disease and that can be readily transmitted from one individual to another, directly or indirectly. Effective treatment and preventive measures are not usually available.

(Mars Sample Return backward contamination–Strategic advice and requirements : 23)

Cassie Conley, former NASA planetary protection officer from 2006 - 2018 summarized their policy at the time like this in an official NASA video which they have not taken down:

“that means we are going to contain the samples as if they were the most hazardous Earth organisms that we know about, Ebola virus.”

at 1:02 into this official NASA video

But NASA are not following this advice.

Invalid use of ten examples of pathogens that co-evolved with humans or other Earth hosts to conclude that Mars is likely to have near zero probability of a direct pathogen - there are many counterexamples of pathogens that didn't co-evolve with humans or any other Earth host, like tetanus or Aspergillus fumigatus

The Biological Safety Report argues that there is near zero probability of a risk group 4 organism or indeed any pathogen of humans. The way they reason is that they look at ten examples of human diseases (Ebola, HIV, influenza, Escherichia coli strain 0157:H, Candiasis yeast, Kaposi's sarcoma, malaria, yellow fever, and schistosomiasis).

They find that all these diseases needed to evolve in an Earth host, they either co-evolved with humans or jumped to humans from birds or mammals, or in the case of malaria, yellow fever and schistosomiasis, evolved in a more complex relationship between humans and snails or mosquitoes (with common ancestor between snails or mosquitoes and humans 600 to 1,200 million years ago).

Biased on these examples they conclude that we have near zero risk from Martian microbes

“Since any putative Martian microorganism would not have experienced long-term evolutionary contact with humans (or other Earth host), the presence of a direct pathogen on Mars is likely to have a near-zero probability.”

(Craven et al., 2021, Biological safety : 6)

This passage has no cites to the planetary protection literature. They don't say that anyone else has used this argument before. The only previous occurrence of this argument I know of is the non peer reviewed op ed by Robert Zubrin.(more below)

Warmflash et al looks at various counterexamples (Warmflash et al, 2007, Assessing the Biohazard Potential of Putative Martian Organisms for Exploration Class Human Space Missions : 14 - 15)

• Legionnaire's disease
• Tetanus (caused by Clostridia tetani)
• Clostridia perfringens, one of the most common causes of food poisoning

The Clostridia genus is anaerobic so doesn't need oxygen. They also look at diseases of crops that can harm humans when they eat them

• Botulism (C. botulinum)
• Ergot disease (C. purpurea)

As we've seen above we also now have an example of a Mars analogue black yeast (fungus) that does well in Mars simulation chambers

• The NRC study dates from 14 years ago and a new study would find examples of Mars analogue organisms that can be human pathogens such as the black yeast Exophiala jeanselmei MA 2853 which is found in rocks in Antarctica as well as in warmer conditions, and can grow up to human blood temperature, risk group 2 (moderate individual risk, low community risk)

I used that to motivate my illustrative worst case of a novel genus like Aspergillus which is not adapted to humans either and has severe effects every year - Aspergillus does require oxygen but can manage in low oxygen conditions in the lungs. Mars might actually have oxygen rich very cold brines by some recent research - or it could be a microbe that doesn't need oxygen at all but otherwise an analogue

• Illustrative worst case scenario for human health and wildlife (especially birds):
  - NOVEL GENUS OF FUNGI from Mars able to infect us similarly to Aspergillus fumigatus
  - harms 200,000 immunocompromised a year, with high fatality rate 30% to 95%
  - harms 48 million with an allergic lung disease (ABPA, 2.5% of asthma cases)
  - harms 400,000 with a serious chronic allergic lung disease
  - and not adapted to an infectious lifestyle in any organism
  [NEW detailed scenario, fungal pathogens IN NEPA BOTH]

Warmflash et al conclude:

While, based on the terrestrial examples, invasive capabilities will likely be rare among putative Martian microorganisms (32), we cannot be sure that they will be non-existent, nor can we depend on the following a priori conclusion, as expressed by a popular Mars colonization enthusiast, that there is no evidence for the existence of macroscopic Martian fauna and flora. Without indigenous hosts, the existence of Martian pathogens is impossible (50). In fact, not even all infectious human pathogens - let alone non-infectious pathogens - on Earth require a multicellular, macroscopic host in order to evolve harmful capabilities.

(Warmflash et al, 2007, Assessing the Biohazard Potential of Putative Martian Organisms for Exploration Class Human Space Missions : 14 - 15)

But the biological safety report show no awareness of these examples.

See above: In worst case scenarios microbes do NOT need to have long-term evolutionary contact with Earth hosts to harm us - example of tetanus which kills thousands of unvaccinated newborns every year and legionnaires disease which is a disease of protozoa and biofilms not adapted to invade lungs which also kills

Indeed, the Biological Safety Report gives no cites to the planetary protection literature for this paragraph discussing human diseases although it has of course been one of the main topics of discussion for decades. Also they don't cite any previous cite to any paper arguing that microbes that haven't co-evolved with Earth hosts can't harm us.

The only paper I know of that uses anything resembling this argument is the non peer reviewed Op. Ed. by space colonization enthusiast Robert Zubrin, the author mentioned by Warmflash et al. as their cite (50)

But couldn't such life, if somehow unearthed by astronauts, be harmful? Absolutely not. Why? Because disease organisms are keyed to their hosts. Like all other organisms, they are specially adapted to life in a particular environment. In the case of human disease organisms, this environment is the interior of the human body or of a closely related species, such as another mammal. For almost 4 billion years, the pathogens that afflict humans today have waged a continuous biological arms race with the defenses developed by our ancestors. An organism that has not evolved to breach our defenses and survive in the microcosmic free-fire zone that constitutes our interiors will have no chance of successfully attacking us. This is why humans do not catch Dutch elm disease and trees do not catch colds. Any indigenous Martian host organism would be far more distantly related to humans than are elm trees.

(Zubrin, 2000, Contamination From Mars: No Threat)

That's rebutted by the response in the next edition of the Planetary Report (the monthly magazine of the Planetary Society) by planetary protection experts (Rummel et al., 2000, Opinion: No Threat? No Way : 4 - 7). But it's widely believed by space enthusiasts who know of Zubrin's article and not the refutation. The biological safety report don't cite Zubrin, which suggests they came up with the reasoning independently, but either way it's invalid reasoning as we saw.

Indeed, the biological safety report give no cites for the ten diseases either to show they co-evolved with humans or other Earth hosts, though it's obvious for most of them, This paragraph has only have one cite, to show that humans have a common ancestor with snails and with mosquitoes 600 million to 1200 million years ago.

On looking closer, it turns out that one of their ten examples is controversial. The strain of e-coli that produces Shiga's toxin may have got that capability in biofilms rather than humans. That's a theory of Łoś et al. from 2013 (Altruism of Shiga toxin-producing Escherichia coli: recent hypothesis versus experimental results, 2013).

Łoś et al. reasoned that:

• e. coli strain 0157:H rarely spreads human to human. It only does this during rare outbreaks
• this strain of e.coli kills itself in humans when it produces Shiga’s toxin – so it only benefits other e. coli altruistically by destroying the white blood cells (phagocytes) that attack them.
• this strain of e. coli stays alive in biofilms when it produces Shiga’s toxin – in biofilms, the toxin only kills the attacking protozoa.

So Shiga’s toxin seems to be more beneficial to e. coli in biofilms than in humans, and human outbreaks don’t spread often enough or far enough for much evolution.

A 2018 review paper by Sun et al. looks at the arguments for and against this suggestion as there is some experimental evidence both ways (Dual role of mechanisms involved in resistance to predation by protozoa and virulence to humans, 2018). They concluded:

In conclusion, evolution of mechanisms that allow for survival within protozoa may have selected for traits that also allow bacteria to escape that harmful effects of phagocytes [in humans].

. Dual role of mechanisms involved in resistance to predation by protozoa and virulence to humans.

Whether or not Shiga's toxin did evolve in a biofilm, the better known example of Legionnaires' disease certainly did, another example covered in the paper by Warmflash et al. (Warmflash et al, 2007, Assessing the Biohazard Potential of Putative Martian Organisms for Exploration Class Human Space Missions). While other examples like Tetanus and Aspergillus fumigatus (which seems to be new to the planetary protection literature) show they don't even need to evolve to infect protozoan hosts to harm humans.

One feels this paragraph got little attention, perhaps there was nobody on the team with the relevant expertise to check it? Or not enough time to check it?

Your first planetary protection officer, John Rummel says people have to have some kind of respect for the unknown

John Rummel, NASA planetary protection officer from 1997 to 2006, as interviewed by Scientific American in 2022 after the first round of public comments on your proposals summarized it like this:

We keep finding Earth organisms doing new things that are quite interesting from the standpoint of potential life elsewhere. So why don’t we think we need to be careful? The answer is that we do need to be careful, as repeatedly emphasized by the National [Academies]...

People have to have some kind of respect for the unknown. If you have that respect, then you can do a credible job, and the public is well-served by your caution.”

(Controversy Grows Over whether Mars Samples Endanger Earth)

General picture from ALL studies back to the Apollo era - it appears to be low risk but we need respect for the unknown
- even a low risk of this nature is highly significant and we do need to protect Earth's biosphere and its inhabitants even from a low risk of large-scale harm

That’s the general picture here from ALL the major reports ever commissioned on the topic back to the Apollo era as we saw (Mars Sample Return backward contamination–Strategic advice and requirements : 20) (Meltzer, 2012, When Biospheres Collide : 35, 420)

It appears to be a low risk but we need a respect for the unknown, and as a result this low risk is highly significant.

Even a low risk of large-scale effects must be considered carefully and thoroughly. I don’t see that in this EIS. There is no mention of the conclusion of previous studies that we have to contain the samples as if they were the most hazardous Earth organisms known.

The final Environmental Impact Statement covers the NEPA requirement to consider all major points of view on environmental effects with a statement that the National Academy of Sciences Mars sample return study in 2009 and Martian moons sample return study in 2019 "affirmed the consensus" that the Mars meteorite argument is valid
- BOTH STUDIES said clearly this argument is INVALID
- no mention of Cassie Conley's statement in an official NASA video that the samples will be treated as the most hazardous Earth organisms known
- no mention of the European Space Foundation recommendation to treat the samples as risk group 4 organisms

NASA have a legal requirement in a draft EIS to summarize all major points of view on the environmental impacts. The draft EIS never mentions that the samples have to be contained as if they were the most hazardous organisms known or that the risk of large-scale harm to human health can't be shown to be non zero. All you say is that

The relatively low probability of an inadvertent reentry combined with the assessment that samples are unlikely to pose a risk of significant ecological impact or other significant harmful effects support the judgement that the potential environmental impacts would not be significant.

(NASA, 2023, MSR FINAL PEIS :3-16)

The draft EIS do cover the views of two National Research Council studies - but they deal with the NEPA requirement with an incorrect summary that says that these previous sample studies are in agreement with their own conclusions.

- NASA's draft EIS MISTAKENLY summarized then National Academy of Science study from 2019 as "Affirming the consensus" for the Mars meteorite argument and didn't notice that it said the meteorite argument is INVALID for samples from Mars

They then incorrectly summarize the conclusions about the Mars meteorite argument which we've seen is invalid: These are the relevant statements in NASA's draft EIS:

One of the reasons that the scientific community thinks the risk of pathogenic effects from the release of small amounts ( less than 1 kilogram [ 2.2 pounds ] ) of Mars samples is very low is that pieces of Mars have already traveled to Earth as meteorites. The National Academies of Sciences affirmed the consensus that Martian material travels to Earth when they developed the planetary protection guidelines for sample return from Martian moons, Phobos and Deimos (National Academies of Sciences, ..., 2019) .

...

“The natural delivery of Mars materials [i.e. martian meteorites that reach Earth] can provide better protection and faster transit than the current MSR mission concept … First, potential Mars microbes would be expected to survive ejection forces and pressure (National Academies of Sciences, …, 2019), …”

(NASA, 2023, MSR FINAL PEIS 3–3),

[my comment in red]

The argument is rebutted by the National Academy of Sciences in the very cite which they say "affirmed the consensus" that the argument is valid, the 2019 guidelines for the Martian Moons

“The sample may well come from an environment that mechanically cannot become a Mars meteorite. The microbes may NOT be able to survive impact ejection and transport through space.”

...

Finding: The committee finds that the content of this report and, specifically, the recommendations in it do not apply to future sample return missions from Mars itself.

(SSB, 2019, Planetary protection classification of sample return missions from the Martian moons : 45)
[CAPS added to highlight the discrepancy]

As we've seen the 2009 cite also said clearly that the Mars meteorite argument is invalid.

Thus, the potential hazards posed for Earth by viable organisms surviving in samples is [are] significantly greater with a Mars sample return than if the same organisms were brought to Earth via impact-mediated ejection from Mars

(SSB, 2009, Assessment of planetary protection requirements for Mars sample return missions : 47)
[See end of section "The survival of organisms ejected from Mars]

As a result of this incorrect summary the EIS don't need to elaborate on any other point of view later in the EIS because they have already said that there is a consensus, that all the other major points of view are in agreement with them. This of course is incorrect as we saw in the previous sections.

It would be more accurate to say there is a consensus that ALL the major peer reviewed literature concludes that not just the meteorite argument is invalid, but ALL of the four main arguments.

• These four plausible but invalid arguments reinforce false beliefs in the space community - which they use in good faith to recommend dropping all planetary protection for Mars and Earth

- NASA's draft EIS didn't notice the NRC in 2009 said hydrothermal vent organisms HAVE COMMON VIRULENCE GENES with relatives that harm humans and that we need to treat Mars samples as having a risk of large-scale harm to human health

This is how the draft EIS summarizes the situation - they give the impression that the NRC in 2009 and 2019 endorsed their conclusion that the pathogenic effects would not be significant

The NRC reaffirmed the conclusion that the potential for pathogenic effects from the release of small amounts of Mars samples is regarded as being very  low. Additionally, those life forms found in extreme environments on Earth have not been found to have pathological effects on humans (National Research Council 2009 ) .

(NASA, 2023, MSR FINAL PEIS :3-16)

In that paraphrase they miss out a very important "however" which leads to the conclusion that we have to treat the samples as pathogenic to humans.

“However, it is worth noting in this context that interesting evolutionary connections between alpha proteobacteria and human pathogens have recently been demonstrated for natural hydrothermal environments on Earth

... it follows that, since the potential risks of pathogenesis cannot be reduced to zero, a conservative approach to planetary protection will be essential, with rigorous requirements for sample containment and testing protocols of life forms that are pathogenic to humans’

(SSB, 2009, Assessment of planetary protection requirements for Mars sample return missions : 46)

The NRC citation is to two species of microbes that live in the hot hydrothermal vents on the sea floor. These are strains of the class epsilon-Proteobacteria, (Deep-sea vent ε-proteobacterial genomes provide insights into emergence of pathogens. ) now reclassified as Epsilonbacteraeota (Comparative genomic analysis of the class Epsilonproteobacteria and proposed reclassification to Epsilonbacteraeota (phyl. nov.) ).

These organisms don’t harm us, but their close relatives can. Helicobacter can cause stomach ulcers and Campylobacter can cause acute gastrointestinal disease in humans (Epsilonproteobacteria in humans). These pathogens harm us through virulence genes they share with the hydrothermal vent organisms. The same adaptations that help them survive in their ecological niches in hydrothermal vents also help them survive in humans

Although they are nonpathogenic, both deep-sea vent epsilon-Proteobacteria share many virulence genes with pathogenic epsilon-Proteobacteria, [they give a list of virulence genes, and other capabilities that enhance virulence] these provide ecological advantages for hydrothermal vent epsilon-Proteobacteria who thrive in their deep-sea habitat and are essential for both the efficient colonization and persistent infections of their pathogenic relatives.

(Deep-sea vent ε-proteobacterial genomes provide insights into emergence of pathogens )

The passage from the NRC about the potential for pathogens of humans concludes that since the potential risk to humans from life returned from Mars can’t be reduced to zero a conservative approach to planetary protection is essential. It's clear here they are talking about large-scale effects on human health.

“TITLE: Types of large scale effects

SUBTITLE: Large scale negative pathogenic effects on humans
…
… It follows that, since the potential risks of pathogenesis [disease causing infection of humans] cannot be reduced to zero, a conservative approach to planetary protection will be essential, with rigorous requirements for sample containment and testing protocols of life forms that are pathogenic to humans.

(SSB, 2009, Assessment of planetary protection requirements for Mars sample return missions : 46)

The NRC study also said that it is not possible to assess future negative impacts caused by delivery of putative extraterrestrial life, based on current evidence

… it is not possible to assess past or future negative impacts caused by the delivery of putative extraterrestrial life, based on current evidence.

... The committee found that the potential for large-scale negative effects on Earth’s inhabitants or environments by a returned martian life form appears to be low, but is not demonstrably zero

: (SSB, 2009, Assessment of planetary protection requirements for Mars sample return missions : 48 ).

A new study would find examples of Mars analogue organisms that can be human pathogens such as the black yeast Exophiala jeanselmei MA 2853 which is found in rocks in Antarctica as well as in warmer conditions, and can grow up to human blood temperature risk group 2 (moderate individual risk, low community risk) - new since 2009

However we have many terrestrial fungi which do well in Mars simulation chambers suggesting in the other direction that it’s a realistic possibility that Mars has fungi that would be able to grow on Earth. Many of our candidates for fungi that might live on Mars are rock inhabiting black fungi. These are able to adapt to extreme environments, hot and cold and other extremes such as high salinity, acidity, and desiccation, and many have been able to colonize rocks in Antarctica (Rock black fungi: excellence in the extremes, from the Antarctic to space., 2015)

One of these black fungi, Cryomyces antarcti was tested in the BIOMEX experiment simulating a Martian atmosphere, exterior to the ISS. At the end of the experiment it was not only still viable but showed only slight damage too fine to see with optical microscopy ( . BIOMEX experiment: ultrastructural alterations, molecular damage and survival of the fungus Cryomyces antarcticus after the experiment verification tests. 2017) These types of fungi have been given many names in the literature including “black yeasts” and “micro-colonial fungi”.

One of these black fungi is closely related to human pathogens. That’s Exophiala jeanselmei MA 2853, a rock inhabiting black fungus in moderate climates, which turned out to have the potential to survive and grow in the Mars simulation chamber of the German aerospace center with daily temperature changes from below -40°C to above 15°C and also simulating the day to night humidity cycle (Protein patterns of black fungi under simulated Mars-like conditions, 2014)

E. jeanselmei is sometimes a pathogen itself and its close relatives are sometimes fatal. In a review of 84 case reports since 1980, 2 out of 29 patients with normal functioning immune system died and 7 out of 55 immunocompromised cases died (Cutaneous Phaeohyphomycosis of the Right Hand Caused by Exophiala jeanselmei:, 2022 : table 2 ). These were all classified at the time as E. jeanselmei, but many early cases may be other cryptic species of Exophiala that are clinically identical and can only be distinguished with gene sequencing such as such as E. heteromorpha, E. lecanii-corni, E. oligosperma, and E. xenobiotica, and . E. jeanselmei itself is also sometimes an opportunistic pathogen of humans ( . Spectrum of clinically relevant Exophiala species in the United States. ). T his for example is a case from 2010 confirmed by gene sequencing (The clinical spectrum of Exophiala jeanselmei, with a case report and in vitro antifungal susceptibility of the species) as was the case studied by Wu et a (Cutaneous Phaeohyphomycosis of the Right Hand Caused by Exophiala jeanselmei:, 2022)

It's classified as a risk group 2 organism (moderate individual risk, low community risk) and can grow up to 37°C but not up to 40°C (University of Adeleide, n.d., Exophiala) [click to expand the Exophiala Jeanselmei section]

The EIS doesn't mention the conclusion of the 2012 ESF study that the samples should be treated as risk group 4 organisms or the conclusion of their former planetary protection officer that NASA will treat them as if they are the most hazardous organisms known:

• 2012 ESF study says Mars samples should be treated as risk group 4 Earth organisms with high individual and community risk, usually no treatment available, until we know more
  - and your former planetary protection officer Cassie Conley says we will treat them as the most hazardous organisms known in an official NASA video

Based on this incorrect summary of the literature and their own conclusions, the draft EIS says they only need to look at the risk of sample release during landing and recovery
- i.e. no need to consider lab leaks
- and they say they are in the middle of a calculation of the risk of harm to humans from release of the samples
- but don't set a target probability that they would consider as an acceptable conclusion of that calculation

NASA then say that as a result of all this they only need to look at the risk of Mars sample release during landing and recovery of the samples

Because it is currently thought the potential for pathogenic effects from the release of small amounts of Mars samples is regarded as being very low, the analysis of Health and Safety in Section 3.4 focuses on the design mitigations and protocols utilized to minimize the potential risk associated with Mars sample release during landing and recovery.

So, by omission, they are saying they don't need to look at the risk of lab leaks. So they don't need to consider the tricky issue of quarantine of human technicians - the word quarantine doesn't occur in the EIS except for one instance historically in connection with Apollo. (NASA, 2023, MSR FINAL PEIS :3-15)

On risk assurance, though NASA don't set a target probability they do say they will try to calculate the potential for pathogenic effects from release of small amounts of the samples.

To assess the risk associated with the return of samples, NASA has identified multiple vectors (specific pathways) that could result in the release of Mars material into Earth’s biosphere. However, a final quantitative estimate of the likelihood of release for any one vector or group of vectors based on the MSR Campaign design and mission plans is not complete, and the assessment of each of these vectors is ongoing.

Because it is currently thought the potential for pathogenic effects from the release of small amounts of Mars samples is regarded as being very low, the analysis of Health and Safety in Section 3.4 focuses on the design mitigations and protocols utilized to minimize the potential risk associated with Mars sample release during landing and recovery.

Should further refinement of mission and design elements result in the potential for substantive impacts outside the scope of those analyzed in this PEIS, then supplemental National Environmental Policy Act (NEPA) analysis may be required.

(NASA, 2023, MSR FINAL PEIS :3-4)

However, they don't define what is meant by "very low" or "substantive" in terms of a probability and don't say what level of risk they consider acceptable as we saw:

• Chester Everline
  - a NASA employee and a co-author of your handbook on probabilistic risk assurance
  - says that he didn't find a target probability
  - and that if the meteorite argument can't be established (it can't as we saw) the EIS should consider an alternative of deferred return
  - not to return the samples until the risks are better understood [NEW]

With this statement that the environmental effects would not be significant NASA's draft EIS achieves a major legal simplification - e.g. no need to consider impacts on the Great Lakes, or oceans, or invasive species and minimal need to involve other agencies - not suggesting for a moment that this is the motivation but it's the effect of it

By saying that environmental effects would not be significant, NASA can skip various presidential directives to consider:

• impact on the environment,
• impact on the oceans,
• impact on the great lakes,
• escape of invasive species,
• lab biosecurity against theft

and many others

In more detail (NASA, 2012, NASA Facilities Design Guide) .

      EXECUTIVE ORDERS

1. 13112 Invasive Species Federal agencies are to prevent the introduction of invasive species, to provide for their control, and to minimize the economic, ecological, and human health impacts that invasive species cause
    
2. 13158 Marine Protected Areas Federal agency actions are to avoid harm to the natural and cultural resources that have been designated a Marine Protected Area (MPA).
    
3. 13186 Responsibilities of Federal Agencies to Protect Migratory Birds In their land management and environmental quality planning, Agencies are to avoid/minimize adverse impacts on and to prevent/abate pollution or detrimental alteration of the environment of migratory bird resources.
   [migratory birds could be affected by environmental changes from introduction of mirror life, or by novel diseases such as a novel fungal genus]
    
4. 13547 Stewardship of the Ocean, Our Coasts, and the Great Lakes Federal Agencies are to exercise stewardship of oceans, coasts, and the Great Lakes by protecting, maintaining, and restoring their health and biological diversity.
    
5. Endangered Species Act Through federal action and by encouraging the establishment of state programs, the 1973 Endangered Species Act provided for the conservation of ecosystems upon which threatened and endangered species of fish, wildlife, and plants depend
   [effects of introduced microbes from Mars on ecosystems would not be possible to predict]
    
6. 13486 Strengthening Laboratory Biosecurity in the United States Facilities that possess biological select agents and toxins are to have appropriate security and personnel assurance practices to protect against theft, misuse, or diversion to unlawful activity of such agents and toxins.
   
   FEDERAL LAW
   
7. Migratory Bird Treaty Act Act for the protection of migratory birds.
    
8. Safe Drinking Water Act This Act regulates drinking water systems
   [microbial life from Mars could potentially have effects on drinking water]
   

This list is not likely to be complete. The authors of that list wouldn't consider the executive orders relevant to, say, effects of releasing life based on mirror organics on the environment.

Then by presenting the potential environmental effects as not significant, they also

• don't need much consultation with other agencies like Public health, CDC, DoA, etc
• no need to discuss quarantine of technicians.

Then with the new fast track NEPA process they can complete it all in a year, giving much less time for others to notice and challenge their plans. Before the NEPA modernization, the average time was 4.5 years and something like this would have likely taken longer.

CEQ found that over the past decade, the average time for agencies to complete an EIS was 4.5 years. CEQ’s current guidance suggests that this process, even for complex projects, should not take more than one year. (NEPA Modernization)

Based on what the European Space Foundation Sample Return study of 2012 says about a low risk of large-scale effects
- this mission also has international implications for ESA member countries, and the EU and also internationally
- including obligations under EU Directive 2001/42/EC, the Espoo convention and the Biodiversity Convention

This is what the European Space Foundation says in the most recent Mars Sample Return study

The Study Group also concurs with another conclusion from the NRC reports (1997, 2009) that the potential for large-scale effects on the Earth's biosphere by a returned Mars life form appears to be low, but is not demonstrably zero. It adds that if this risk appears to be low for free-living self-replicating organisms, considering their specificities and replication requirements, the potential risk posed by virus-type and gene transfer agent-type entities can be considered to be far lower and almost negligible, but still cannot be demonstrated to be zero.

(Ammann et al., 2012, Mars Sample Return backward contamination–Strategic advice and requirements : 20)

Even if the US Environmental Impact Statement says there is no risk of any environmental effects, this is domestic US law and does not apply in EU member states, UK or Canada or of course internationally.

The initial sample receiving facility will be in the USA with the current plans - but ESA member states are involved in returning the samples from Mars to the USA, so they also are involved. Directive 2001/42/EC would apply Directive 2001/42/EC of the European Parliament and of the Council of 27 June 2001 on the assessment of the effects of certain plans and programmes on the environment. This is similar to NEPA, including the need for a draft report, public comments and so on. This is an example of how it is implemented in the UK, which still retains the law though no longer in the EU. (Strategic Environmental Assessment Directive: guidance - Practical guidance on applying European Directive 2001/42/EC)

Also the UK and many other member states of ESA are parties to the Espoo convention (Environmental Assessment - Espoo Convention). Under that convention they need to consult with each other on all major projects that can have an effect outside of their own boundaries - which would certainly apply to a Mars sample return release of life that transformed the biosphere.

Then, though the USA is not part of the Convention on Biological Diversity, all the participating nations of ESA are, indeed all UN nations except the USA which leads to obligations to prevent introduction of alien species that threaten ecosystems, habitats or species:

"Each Contracting Party shall, as far as possible and as appropriate:

...(h) Prevent the introduction of, control or eradicate those alien species which threaten ecosystems, habitats or species."

Convention on Biological Diversity, 1997, Legal text

- this mission also has implications internationally e.g. for the WHO, FAO etc

There are many other relevant conventions and treaties and once the US says there is potential for large scale effects, it would likely involve:

• international organizations like the WHO, FAO etc
• international treaties

No matter which country is involved in planning a Mars sample return mission, at some stage, international agencies like the Food and Agriculture Organization may get involved, because of potential impact on agriculture and fisheries and global food supplies, and the World Health Organization because of effects on human health globally if a new organism is returned that can be spread to other countries.

They might not have much by way of authority but they would affect public opinion in the USA.

There are many international implications. See: (Updating Planetary Protection Considerations and Policies for Mars Sample Return) and (Planetary Protection, Legal Ambiguity, and the Decision Making Process for Mars Sample Return)

This is not suggesting in any way that NASA's motivation was to simplify the international legal situation. But it is an effect of this statement.

All these precautions are there for a reason. We need to be especially careful about a decision that can potentially lead to bypassing all these precautions that protect Earth's biosphere.

NASA plan to use many basic principles of a Biosafety Level 4 facility to contain the samples - this is based on the science of 1999 but is now out of date for a Mars sample return [IN NEPA BOTH]

Video: Open letter to NASA - proposal for BSL-4 is based on science of 1999 - can't stop ultramicrobacteria

NASA plan to contain the samples using many of the basic principles of a Biosafety level 4 facility (BSL-4) facility - based on the science of 1999

This is what NASA say:

Nevertheless, out of an abundance of caution and in accordance with NASA policy and regulations, … NASA and its partners would use many of the basic principles that Biosafety Level 4 (BSL-4) laboratories use today to contain, handle, and study materials that are known or suspected to be hazardous.

(NASA, 2023, MSR FINAL PEIS S-4),

The science of 1999 did set requirements achievable by a BSL-4 – that the probability that a single particle of 0.2 microns or larger is released into Earth’s environment is less than 1 in a million (Mars Sample Return backward contamination–Strategic advice and requirements : 3).

The European Space Foundation in 2012 said we have to contain the far smaller ultramicrobacteria and gene transfer agents
- 100% containment at 0.05 microns or larger and 1 in a million for release of a single particle ever at 0.01 microns or larger
- well beyond capabilities of a BSL-4

The European space foundation said we have to contain ultramicrobacteria and gene transfer agents - well beyond the capabilities of a BSL-4 - based on the science of 2012 - the ESF in 2012 updated this 1999 requirement to 1 in a million containment for a single particle of 0.01 microns or larger, and 100% containment for 0.05 microns or larger (Ammann et al., 2012, Mars Sample Return backward contamination–Strategic advice and requirements : 48).

Screenshots from (Ammann et al., 2012, Mars Sample Return backward contamination–Strategic advice and requirements : 21 and: 48).

Their recommendation of 100% containment at 0.05 microns was the result of reviewing reports that ultramicrobacteria are still viable after passing through a 0.1 micron nanopore (Ammann et al., 2012, Mars Sample Return backward contamination–Strategic advice and requirements : 15). They cited two studies, in freshwater from Greenland (Detection and isolation of ultrasmall microorganisms from a 120,000-year-old Greenland glacier ice core) and 20 different sites in Switzerland (Quantification of the filterability of freshwater bacteria through 0.45, 0.22, and 0.1 μm pore size filters and shape-dependent enrichment of filterable bacterial communities).

This has been confirmed many times since then.

Text on graphic: Size limit 1999 to 2012: 0.2 microns

ESF Size limit (2012): 0.05 microns

The European Space Foundation study in 2012 reduced the limit from 0.2 microns to 0.05 microns after the discovery that these ultramicrobacteria are viable after passing through 0.1 micron nanopores

Next size limits review might reconsider ribocells – theoretical size limit 0.01 microns

ESF limit = ~⅛ of the wavelength of violet light

Background graphic: SEM of a bacterium that passed through a 100 nm filter (0.1 microns), larger white bar is 200 nm in length (Passage and community changes of filterable bacteria during microfiltration of a surface water supply)

violet bar for shortest wavelength of violet light (380 nm or 0.38 microns)

Mars might well have tiny microbes because

• very small cells can escape grazing by larger grazing amoebas which don’t notice them
• very small cells have a larger surface area to volume ratio, so can use nutrients better in nutrient poor conditions

See: (Ghuneim et al., 2018, (Nano-sized and filterable bacteria and archaea: biodiversity and function)

Their 1 in a million containment at 0.01 microns or larger is because of a discovery that the even smaller Gene Transfer Agents can transfer genes and so, genetic capabilities such as antibiotic resistance to the genomes of distantly related microbes (archaea) in sea water far more rapidly than previously thought, overnight (Ammann et al., 2012, Mars Sample Return backward contamination–Strategic advice and requirements : 19) citing (High frequency of horizontal gene transfer in the oceans) (summarized in (Virus-like particles speed bacterial evolution))

I alerted you to this issue that a BSL-4 can’t meet the requirements of the ESF study in the first round of public comments

This 100% containment at 0.05 microns is well beyond capabilities of BSL4 facilities. Even ULPA level 17 filters only contain 99.999995 percent of particles tested only to 0.12 microns (BS, 2009:4).

...

An experimental 6-layer charged nanofiber filter intended for coronaviruses filters out 88% of ambient aerosol particles at 0.05 microns (Leung et al, 2020).

Even this would not achieve 100% containment.

… (Comment posted on May 16th by Robert Walker to NASA’s first request for comments on their plans)

We don't need to contain ultramicrobacteria in terrestrial labs or gene transfer agents - and nobody seems to be working on filters to do this - but ultramicrobacteria could bring novel life and gene transfer agents could bring novel genetic capabilities to Earth for related life

Nobody seems to be working on the technology to contain ultramicrobacteria or GTAs - we don’t normally need to do this. Modern reviews of air filter technology don’t mention any attempts to achieve such capabilities (Application of Electrospun Nonwoven Fibers in Air Filters).

Ultramicrobacteria could bring novel life (such as mirror life or other exotic biology) to Earth from another biosphere and if Martian life is related to terrestrial life, gene transfer agents could bring novel capabilities to terrestrial microbes that Martian life evolved in the very different conditions on Mars.

The ESF says we need to do a new review of the size limit and level of assurance regularly - definitely needed a decade later - so even their limit is now out of date

Also before developing any such technology we need a review of the size limits and level of assurance - the ESF in 2012 said we need to do this regularly

Based on our current knowledge and techniques (especially genomics), one can assume that if the expected minimum size for viruses, GTAs or free-living microorganisms decreases in the future, and this is indeed possible, it will be at a slower pace than over the past 15 years.

However, no one can disregard the possibility that future discoveries of new agents, entities and mechanisms may shatter our current understanding on minimum size for biological entities. As a consequence, it is recommended that the size requirement as presented above is reviewed and reconsidered on a regular basis

(Ammann et al., 2012, Mars Sample Return backward contamination–Strategic advice and requirements : 21)

RECOMMENDATION 8: Considering that (i) scientific knowledge as well as risk perception can evolve at a rapid pace over the time, and (ii) from design to curation, an MSR mission will last more than a decade, the ESF-ESSC Study Group recommends that values on level of assurance and maximum size of released particle are re-evaluated on a regular basis

(Ammann et al., 2012, Mars Sample Return backward contamination–Strategic advice and requirements : 48)

A decade later this review is needed first before we can design any air incinerators or filters to the new standard.

A new size limit review could look at recent research into the very tiny ribocells

The next size limit review may examine new research into extremely small early life cells such as ribocells with enzymes made from fragments of RNA instead of proteins (Kun ,2021.Maintenance of Genetic Information in the First Ribocell). Steven Benner and Paul Davies say the small 0.01 micron diameter structures in the martian meteorite ALH84001 are consistent with RNA world cells (Towards a Theory of Life : 37).

Panel 4 for the 1999 workshop estimated that one of the structures in the Mars meteorite ALH84001 was consistent with a minimum volume early life RNA world cell 14 nm in diameter and 120 nanometers in length, if there is an efficient mechanism for packing its RNA (SSB, 199, Size limits of very small microorganisms: proceedings of a workshop : 117). New research into ribocells might lead to a review panel to reconsider this suggestion.

A new size limit review could look at prions - raised by the biosafety report and last considered in 1997 because of the very recent discovery of prions in all three of the domains of life, archaea, bacteria and eukaryotes and so probably in the last common universal ancestor too [NEW]

[At the time of the last public comment I assumed that prions are harmless as they aren't mentioned in any of the recent Mars sample return reviews - but from my preliminary literature survey this might need to be reviewed again by experts as a result of the recent discovery of prions in all three of the domains of microbial life, most recently in the archaea. This wasn't known when prions were last assessed in the planetary protection literature in 1997]

The next size limit review might also need to look at prions. This a new issue raised by the Biological safety report (Biological safety: 6) that was previously mentioned in the 1997 report but not considered since then (SSB, 2009, Assessment of planetary protection requirements for Mars sample return missions : 60). The 1997 report said:

Subcellular disease agents, such as viruses and prions, are biologically part of their host organisms, and an extraterrestrial source of such agents is extremely unlikely.

(SSB, 1997, Mars Sample Return: Issues and Recommendations : 21)

Biological safety report said most likely Martian prion assemblies are harmless to Earth organisms - but that we need to be able to sterilize them - so which is it?

The Biological safety report argues in the same way that most likely Martian prions are harmless to Earth organisms, unless the terrestrial host was similar to Martian hosts and they argue that Earth is unlikely to have similar hosts

It is most likely that any hypothetical Martian prion or similar protein assembly (if such a prion exists and is present in the cached samples) would be incapable of propagation owing to the lack of available hosts, unless these protein assemblies were essentially similar to human, animal, plant or other Earth analogues, and due to the presence of differing environmental conditions on Earth.
(Craven et al., 2021, Biological safety : 4).

However they also recommend that samples are sterilized sufficiently to inactivate prions before they are released

Likewise, it is not reasonable to focus other efforts specifically on the prions that cause transmissible spongiform encephalopathies such as Creutzfeldt-Jakob Disease in humans (as they are entirely based on misfolded human or animal pro teins), but it is reasonable to focus on inactivation methods that affect protein assemblies similar to prions and the means of fully denaturing (inactivating) those protein polypeptides

(Craven et al., 2021, Biological safety : 11).

If it is indeed necessary to sterilize prions, or at least "protein assemblies similar to prions" to make samples safe for distribution, it may be necessary to contain prions.

So which is it, do we need to contain prions or not? Though they don't mention this in the report, and may not know about it, there is a new factor here not considered in previous reports. We now know that microbes get prion diseases too.

We now know that yeasts, bacteria and archea all get prions - so the last universal common ancestor may well have had prions - and a protein from the bacteria that causes botulism can act as a prion in the unrelated bacteria e. coli

Yeasts often get prions and indeed most of them are beneficial (How do yeast cells contend with prions?.: table 1) but two of them are harmful and yeasts protect against them with various mechanisms (How do yeast cells contend with prions?: figure 2). These are all ones that arise spontaneously in yeast cells. We might find yeasts on Mars especially if it has related life. Some fungi especially black yeasts do well in Mars simulation experiments (BIOMEX experiment: ultrastructural alterations, molecular damage and survival of the fungus Cryomyces antarcticus after the experiment verification tests).

Then, in a discovery from 2017, bacteria such as e. coli can get prions too and in this case a protein Rho from an unrelated bacteria Clostridium botulinum, the microbe that causes botulism, can act as an inheritable prion that changes the functionality of E. coli. They found two such fragments, the shortest of them, a fragment of Rho only 68 amino acids long (A bacterial global regulator forms a prion).

So that also is an example of a prion that comes from an unrelated bacteria°C. botulinum infecting e. coli

Prions are often beneficial to bacteria - but what is beneficial to bacteria might not be so beneficial to their hosts - so are they a concern or not?

The roles of prions are still being unraveled. Prions seem to be rarely harmful in bacteria and frequently beneficial and may help bacteria to cope with stress (Protein aggregation in bacteria : conclusion). However making the bacteria better able to cope with stress could make them more infectious in hosts.

Prions were also found in Archaea in 2021, which means they are now found in all three of the domains of life, Eukarya, Bacteria and Archaea and were likely present in the Last Universal Common Ancestor of all terrestrial life. (The hunt for ancient prions: archaeal prion-like domains form amyloid-based epigenetic elements) which would seem to suggest a high chance of prions on Mars.

From these results, prions from an unrelated bacteria from Mars could in principle make a terrestrial microbe fitter - would that make it more harmful? The prions might be small, with an example of 68 amino acids. It might be necessary to look closer at this, to look at whether prions from Mars could

• change the toxicity of terrestrial microbes, or
• harm terrestrial microbes

So it seems the Biological Safety Report was right to raise the issue of prions again, for the first time since 1997 and to recommend sterilization levels that would destroy them, at least until we know more. A review would need to look at what effects this could have on other organisms and the biosphere, if any.

This may very well conclude that there are no issues. However, given how very small prions are and hard to sterilize, it does seem to be an issue to look at, in a thorough study of the minimum size requirements.

Before we return unsterilized samples we need a size limit review - but before that we need an updated sample return review or both together

If we are going to return unsterilized samples to Earth, we need to do the size limit review first

We can’t develop a filter and / or air incinerator technology and relevant testing requirements, as requirements could change as a result.

But before we can do a size limit review we need to do an updated Mars sample return review (or do both together) so we know what to contain

Science has moved on hugely in the last 14 years since 2009
- many new discoveries e.g. very recent discovery through gene sequencing of "dark matter fungi"
- fungi that infect microbes including fresh water algae
- and for marine algae. a "mystery yet to unravel"
- many topics to consider in a new sample return study in my preliminary survey of the literature

Science has moved on so much in the last 14 years NASA surely needs a new Mars sample return review in 2023 similar to the ones from 2009 (Assessment of planetary protection requirements for Mars sample return missions) and 1997 (Mars Sample Return: Issues and Recommendations) 12 years before the 2009 study.

In my literature survey, I found there have been many advances in science relevant to a Mars sample return since 2009. That includes new discoveries of extremophiles including ones that also do well in normal conditions, capabilities of terrestrial organisms in Mars simulation chambers, new discoveries about terrestrial pathogens such as the very recent discovery of fungal parasites of blue-green algae (Discovery of dark matter fungi) (Basal parasitic fungi in marine food webs—a mystery yet to unravel), new potential habitats on Mars, new discoveries about the potential for life to be transferred in the Martian dust storms, advances in synthetic biology, new ideas about the potential for independently evolved life, and many other topics. See below:.

• Topics that need to be covered in a future Mars sample return backwards contamination study (not likely to be a complete list)

We surely need a new Mars sample return review before we can consider returning unsterilized samples to a receiving lab on Earth - like the study 14 years ago in 2009 and the earlier one 12 years previously in 1997 - each time we find more things that need to be considered

A new review is surely needed before NASA can be ready to do a new EIS, if the mission is going to return unsterilized samples to Earth. The 2012 ESF study focuses mainly on limits of size and filter requirements, and levels of assurance and is itself 10 years out of date now.

Planetary protection officer John Rummel's image of a train wreck for the permitted levels of terrestrial contamination of the samples
- one of the last things your planetary protection experts warned you about before you closed down the planetary protection subcommittee
- risk of returning samples of no astrobiological interest [NEW]

[I discovered John Rummel's comment from 2017 only after the comment process ended but I drew your attention to the issues with terrestrial contamination independently in attachments to the last comment I made see: (Walker, 2022, So many serious mistakes in NASA's Mars Samples Environmental Impact Statement it needs a clean restart : 185 - 194)]

In 2017, your first planetary protection officer John Rummel referred to issues with your permitted levels of terrestrial contamination of the samples as a potential bureaucratic “train wreck”

If the issues aren't resolved, Rummel says, the rover could be headed for a bureaucratic "train wreck".

(Voosen, 2017, With planetary protection office up for grabs, scientists rail against limits to Mars exploration)..

This was one of the last things the Planetary Protection Subcommittee mentions just before they were disbanded: issues with terrestrial contamination of the Mars rover sample tubes. This is in the Planetary Protection Subcommittee report to the NASA Advisory Council, see: (NAC Science Committee, July 25-27, 2016: 9). For brief description of the context see (Space Studies Board, 2018, Review and Assessment of Planetary Protection … : 26–7)

Engineers worried that a sterile sample collection system would have to be transported to Mars in a separate bag
- which might not open - a potential mission critical vulnerability

We do have the technology to make 100% sterile sample tubes free of any organics and to do the same for the sample collection tools. There are various ways to do this including baking the tubes and tools in an oven. However, engineers need a way to protect the tubes and tools from recontamination until after launch. The easiest way to do that is to put them in a bag to keep them sterile, but engineers worried this risks jeopardizing the mission, if Perseverance got to Mars and then they found that the bag couldn’t be opened

On the surface, the answer appears simple—put the sample collection tools inside an air-tight bag and transport it to Mars, keeping the material from ever coming in contact with Earth's atmosphere. But such a solution comes with its own problems.

"The engineers were very worried about this," Sessions said. "Imagine getting to Mars, and you can't get the bags open."

(How Much Contamination is Okay on Mars 2020 Rover?).

NASA went with the engineers here rather than the planetary protection advisors - in the process they achieved a mission with one less critical failure point - but sadly - one likely of virtually no interest for astrobiology

The Perseverance samples will almost certainly be valueless for past life searches (and likely for present-day life too)
- 8.1 ppb of terrestrial organics seems low compared to 100 ppb until you realize that the ionizing radiation leaves < 0.1 ppb of recognizable organics
- astrobiologists want to send instruments to Mars able to detect a single amino acid in a gram
- [IN NEPA BOTH - the < 0.1 ppb figure is NEW]

[The 0.1 ppb estimate is from research in 2022 that turned up as a result of my literature survey]

The permitted levels of organics likely seem low to engineers and geologists:
 

• 8.1 ppb forward contamination by terrestrial organics, 0.7 per biosignature (Mars 2020: mission, science objectives and build : table 6) - this is what they think they achieved. The target was 10 ppb total and 1 ppb per biosignature.
  


(N.B. the probability for a viable organism is per sample tube, their estimate implies a 0.02% chance of finding a single viable terrestrial organism in at least one of the 38 tubes).

However:

• NASA's rocks may have up to 100 ppb of organics, but nearly all of that gets sliced into pieces by ionizing radiation, and the oxidizing effects of activated silica, which then recombine to the extent that the original structure of the molecules is unrecognizable. Consists almost entirely of small chain acids and the original composition is unrecognizable.
   
• ≪ 0.1 ppb of recognizable past amino acids in even recently exposed surface organics on Mars after degradation by ionizing radiation (Pavlov et al., 2022, Rapid Radiolytic Degradation of Amino Acids in the Martian Shallow Subsurface: Implications for the Search for Extinct Life : 111)
   
• This happens with surface exposure ages younger than the youngest exposure age measured by Curiosity - and Perseverance can’t measure exposure ages (Pavlov et al., 2022, Rapid Radiolytic Degradation of Amino Acids in the Martian Shallow Subsurface: Implications for the Search for Extinct Life : 111–112)
   
• Most of the organics with young exposure ages decomposed to: formate \(HCO^{-}_{2}\), oxalate \(C_{2}O_{4}^{2-}\) and some benzoate \(C_{6}H_{5}OO^{-}\) (Pavlov et al., 2022, Radiolysis of macromolecular organic material in Mars-relevant mineral matrices)
e limit for contamination of the returned samples themselves, not just say the walls of the sample tubes(.

Note, NASA is talking about thoroughly mixed contamination here. 8.1 ppb / 0.7 ppb throughout the samples, not just some contamination on the walls of the sample tubes.

The origins of NASA's 10 ppb permitted contamination per biosignature
- their Organic Contamination Panel in 2014
- based on a 10 to 1 signal to noise ratio for measurements of Martian meteorites for the most abundant amino acids
- however the martian meteorites were ejected from meters below the surface of Mars
- and also had less oxidants than we expect from surface rocks
- they did take account of ionizing radiation
- but didn't take account of the effect of ionizing radiation on surface rocks in the presence of oxidants

NASA did comply with requirements set by NASA's iMost team in 2017. This in turn was based on the Organic Contamination Panel from 2014. They used the levels of organics in Martian meteorites to set the limits NASA now use. They tried other lines of evidence but this was the only one they had reasonable confidence in.

The main issue with this is that martian meteorites aren't exact analogues of surface rocks because they were ejected from at least three meters below the surface on Mars. The Organic Contamination Panel only looked at this briefly and didn't have access to the 80 million years exposure age measured by Curiosity.

The limits for Earth-sourced organic carbon in/on the samples originated from a nearly year-long study by a science/Planetary Protection team (OCP) carried out in 2014 (2). Their final recommendation was organized into three components: <1 ppb Tier 1 compounds (organic compounds that would be deliberately evaluated in returned samples as input to life-related interpretations), <10 ppb other C-compounds (everything that is not on the Tier 1 list), and <40 ppb total organic carbon (TOC).

(Beaty et al., 2018, iMOST : 176)

This in turn was based on the deliberations of the Organic Contamination Panel in 2014 (that's the year long study they refer to in the quote).

The Organic Contamination Panel only briefly took account of the effect of ionizing radiation on the levels of biosignatures in the samples.

They say:

On the other hand, martian meteorites arriving on Earth were likely blasted into space from well below the martian surface, where any organics present would have been shielded from the highly oxidizing and radiolytic surface environment. While it is true that near-surface exposure to cosmic radiation likely degrades organic molecules, recent work on Curiosity shows that at least some locations on Mars are eroding fairly rapidly (Farley et al., 2013). Such locations are likely targets for sample collection.

(Simmons et al., 2014, Planning Considerations Related to the Organic Contamination of Martian Samples and Implications for the Mars 2020 Rover : 36)

Their cite is to the Farley et al cite from 2013 / 2014 (Farley et al., 2014, In situ radiometric and exposure age dating of the Martian surface)

However they didn't have access to more recent work on the rapid radiolytic degradation of even such young samples (Pavlov et al., 2022 . Rapid Radiolytic Degradation of Amino Acids in the Martian Shallow Subsurface: Implications for the Search for Extinct Life : 1100 – 1101).

They looked at the likely levels of organics from infall from space and estimated it as 0.2 to 2 ppb for a 100 meter mixing depth or 20 to 200 ppb per biosignature for a 10 meter mixing depth, based from calculations by Benner et al in 2000.

They say most of these organics would be oxidized but that it would be possible to measure the oxidation products (Simmons et al., 2014, Planning Considerations Related to the Organic Contamination of Martian Samples ... : 40).

They looked at the in situ detection of organics by Viking, Phoenix and Curiosity but there is a significant limitation here that none of these instruments could measure levels of large molecules of interest in situ on Mars because they break them up into smaller molecules using pyrolysis (heating in absence of oxygen) to turn them into volatiles that can be analysed by the mass spectrometer. (Simmons et al., 2014, Planning Considerations Related to the Organic Contamination of Martian Samples ... : 39).

They said:

Finding #10: Because we fundamentally do not know what organics would be present on Mars, it is currently impossible to precisely determine what levels of contamination would be necessary in returned samples. There is thus significant uncertainty (in both directions) associated with the proposed limits.

(Simmons et al., 2014, Planning Considerations Related to the Organic Contamination of Martian Samples and Implications for the Mars 2020 Rover : : 29)

They suggested three ways to set a threshold

• Signal to noise ratio, the issue is that they don't know the compounds we expect to find or the lower limits of their concentrations
• Detection limits of the instruments used, this however would require 100% clean samples as some instruments have single molecule detection limits
• Levels of cleanliness that can reasonably achieved for constructing sampling hardware. This could mean developing new technology to achieve those levels.

(Simmons et al., 2014, Planning Considerations Related to the Organic Contamination of Martian Samples and Implications for the Mars 2020 Rover : 29)

For the signal to noise ratio, they based this on an analysis of the organics in our Martian meteorites such as Tissint meteorite, and carbonaceous chondrites, the organics in Murchison meteorite.

(Simmons et al., 2014, Planning Considerations Related to the Organic Contamination of Martian Samples and Implications for the Mars 2020 Rover : tables 6 and 7)

They found that molecules of interest in the martian meteorites ranged from 1 to 100 ppb (nanograms per gram). Based on that they believed that setting a limit of 1 ppb would mean that many of the molecules are detectable against the noise of terrestrial contamination.

Based on the evidence discussed above, our best estimate for concentrations of the most abundant organic molecules of interest (i.e., Tier-I compounds) in returned martian rocks is in the range of 1-100 ng/g. We thus believe that these compounds would likely be measurable above background contamination comprising <1 ng/g per compound. Such background levels should be readily achievable given current technology, and would be at the low end of what is measurable by current survey analytical techniques, thus protecting their role in initial characterization of returned samples. We therefore propose a maximum limit for Tier-I compounds, on a mass/mass basis in returned samples, of 1 ng/g (i.e. < 1ppb).

It is of course possible that analyte concentrations in the returned samples may turn out to be lower than expected, and so the odds of scientific success would be improved by still lower contamination limits. Moreover, lower background levels would permit more accurate and precise measurements at any concentration. Nevertheless, given currently available evidence, it is hard to build a case that contamination limits substantially lower than this would be required to meet either scientific or planetary protection objectives.

(Simmons et al., 2014, Planning Considerations Related to the Organic Contamination of Martian Samples and Implications for the Mars 2020 Rover : 42).

So they proposed a limit that would achieve a 10 : 1 signal to noise ratio.

They agree that it's inevitable many compounds of interest in the samples would be present at much lower levels, but most of the contaminants would also be present at significantly less than 10 ppb.

A frequent point of discussion for the OCP was “What happens to compounds that are less abundant in the returned samples than those discussed above?”.

Certainly it is inevitable that many compounds would be present at <1.0 ng/g. However, setting a contamination limit of 1 (or 10) ng/g does not imply that every compound in Tier I (II) would be present at that level. Rather, sampling surfaces would be cleaned until the most abundant contaminant meets that level, and most other compounds would then be present at much lower levels. The panel thus believes that this strategy represents a sensible compromise, providing reasonably achievable goals while at the same time ensuring that the vast majority (though not necessarily all) analytes of interest would be measurable

(Simmons et al., 2014, Planning Considerations Related to the Organic Contamination of Martian Samples and Implications for the Mars 2020 Rover : 42).

For the total organic carbon they expect concentrations of up to 10 ppm (micrograms per gram) and setting a total organic contamination level of 40 ppb gives a signal to noise ratio of more than 100 : 1. (Simmons et al., 2014, Planning Considerations Related to the Organic Contamination of Martian Samples and Implications for the Mars 2020 Rover : 49).

The issue here is that the martian meteorites were ejected into space from at least 3 meters below the surface, so the levels of organics in those meteorites are not representative of what we might find on the surface, exposed to high levels of ionizing radiation for at least 80 million years for Curiosity's youngest samples:

See:

• (MORE DETAILS - BECAUSE MANY BELIEVE THIS INVALID ARGUMENT)
  our martian meteorites left Mars at least hundreds of thousands of years ago
  - all come from at least 3 meters below the surface
  - modeling suggests they all came from a depth of at least 50 meters
  - many microbes that live in rocks need access to sunlight
  - also many microbes are not adapted to live inside rocks
  - also most of the martian subsurface is very uninhabitable
  - uniform -73°C at a depth of 12 centimeters or deeper
  - except at geothermal hot spots if any

Our martian meteorites that left Mars most recently spent less than a million years traveling through space and none of them have spent significantly more than 20 million years. They also have less oxidants than surface materials which makes them more resilient to ionizing radiation.

They considered in situ measurements of organics, but the data wasn't good enough to set a limit based on measurements from Mars itself because the organics are decomposed into smaller molecules using pyrolysis (heating without oxidation) as part of the processes of analysis used so far (Simmons et al., 2014, Planning Considerations Related to the Organic Contamination of Martian Samples and Implications for the Mars 2020 Rover : 40)

For detectable levels by present day instruments, they found that it wasn't practical to consider detection limits because of ultrasensitive instruments and so they looked at instruments suitable for initial surveys, finding a range of 0.1 to 10 ppb, and selected 1 ppb as a representative value. (Simmons et al., 2014, Planning Considerations Related to the Organic Contamination of Martian Samples and Implications for the Mars 2020 Rover : 41)

As for achievable levels of contamination they were not able to answer this. Geologists don't attempt ultra clean samples but rather reduce levels of organics to be low enough for the measurements they want to make. So they didn't use this criterion (Simmons et al., 2014, Planning Considerations Related to the Organic Contamination of Martian Samples and Implications for the Mars 2020 Rover : 41-2)

So they used the signal to noise ratio to reach their conclusion

That lead to their conclusion

Major Finding #13: We propose the following limits for organic contamination of geological samples by specific compounds: 1 ng/g for Tier-I compounds deemed as essential analytes for mission success, and 10 ng/g for Tier-II compounds (all others)

(Simmons et al., 2014, Planning Considerations Related to the Organic Contamination of Martian Samples and Implications for the Mars 2020 Rover : 43)

With this level of contamination we would detect organics from Mars but it seems unlikely we detect the small trace amounts left after the combination of ionizing radiation and oxidants unless we can find samples with exceptionally young exposure ages.

What we need to prevent in 2033: NASA like the Titanic headed for an iceberg
- when scientists and the public realize NASA has no scientifically credible plans to protect Earth
- and has permitted levels of terrestrial contamination that likely make its samples of virtually no interest for astrobiology past or present [Issue of forward contamination IN NEPA BOTH, metaphor NEWt

John Rummel’s image) brought to mind the iceberg that sunk the Titanic. Here the iceberg represents public and scientific opinion in 2033 when they find out that

• NASA hasn’t prepared scientifically credible plans to protect Earth and your plans weren't prepared with the help of the CDC, NIH, HHS, DoA etc and
   
• the samples you return will likely be valueless for the past life searches (and probably for present-day life too) because of the permitted levels of terrestrial contamination.

Text on graphic: What we need to prevent in 2033 - NASA’s mission plans - NASA’s future science credibility - Public and scientific opinion - No scientifically credible plans to protect Earth - Samples likely valueless for astrobiology because of permitted contamination by Earth life

(Stöwer Titanic)

(NASA logo )

No way to reliably distinguish terrestrial and martian biosignatures
- the attempt at a genetic inventory turned up 1196 distinct genera with too few reads to identify them
~and amongst the 54 genera that could be identified
- four species with less than 98.7% resemblance to the closest known species

We currently have no way to reliably distinguish terrestrial from potential martian biosignatures. We could recognize familiar life like chroococcidiopsis which we already cultivate and have already sequenced. However, the vast majority of microbial species haven’t been characterized or sequenced or cultivated in the laboratory; the problem of “microbial dark matter” (The search for microbial dark matter).

The iMost team suggested a genetic inventory:

To appropriately interpret evidence for Mars life in returned samples, we must be able to distinguish between terrestrial contaminates and indigenous martian life. For this reason, a genetic inventory of both the spacecraft and sample processing/analysis facilities is critical … A genetic inventory represents an important part of the background information related to detection of genetic material in Mars spacecraft and returned samples.

(Beaty et al., 2018, iMOST : 94)

However the issue of microbial dark matter makes it impossible to achieve this goal. Hendrickson did an attempt at a genetic catalogue using 98 swabs from the floors of the clean room that would be used to assemble the Perseverance rover before assembly started. However,

• of 54 identified genera, only 8 were spore forming
   
• Out of 1250 genera, only 54 had enough reads to be identified properly (using the 16s subunit of the ribosome). That leaves 1196 distinct but unidentified genera detected in those 98 swabs.
   
• 36 out of 49 spore forming species (not genera) were only found in one of the 98 swabs 
   
• 4 of the identified species weren’t close to any known terrestrial species (< 98.7% similarity of the 16s subunit to the most similar relative).
  (Hendrickson et al., 2021, Clean room microbiome complexity impacts planetary protection bioburden)

So, 1196 genera were known to be present but couldn’t even be identified. This is not unusual, indeed this is expected and normal.

They used the 16S RNA ribosome subunit for identifying microbial dark matter as it is very stable, and is the basis for the modern classification method for microbes and other organisms due to Carl Woese (The singular quest for a universal tree of life). It is a short section of RNA which gets mixed with proteins to make up the structure of the ribosome used to translate RNA into proteins.

We are certain to detect many novel sequences like this after taking them to Mars and back. So we will get many genetic sequence false positives, and it will be impossible to prove they aren’t martian.

Also the life would have to be very abundant to spot it. A typical sample will be 10 to 15 grams. A 15 gram sample at 8.1 ppb is over 120 nanograms of material (The Sampling and Caching Subsystem (SCS) for the scientific exploration of Jezero crater by the Mars 2020 Perseverance rover : 39-40)

Total Organic Cleanliness of a Sample: Each sample in the returned sample set has less than 10 ppb (parts per billion) baseline or 40 ppb threshold of total organic carbon. Here, 10 ppb baseline can be interpreted as the desired requirement while 40 ppb threshold is the not to exceed requirement. For a 15 g core sample, this equates to 150 ng baseline and 600 ng threshold.

(Mars 2020: mission, science objectives and build : table 6)

One part per billion by mass is the same as one nanogram per gram.

Text on image: Example of how design decisions for Perseverance were based on engineering and geology rather than astrobiology.

This tube was used to collect the first sample from Mars.

For a geologist, it is exceptionally clean, at most 8.1 nanograms of organics and at most 0.7 nanograms per biosignature.

For an astrobiologist, 0.7 nanograms per biosignature is enough to fill at least 7,000 ultramicrobacteria with just that biosignature, e.g. glycine, or DNA (maximum volume 0.1 cubic microns per ultramicrobacteria)

Astrobiologists need 100% clean sample containers with no organics. Their life detection instruments designed for in situ searches on Mrs can detect a single amino acid in a gram.

For engineers, sterilization would add an extra mission critical failure point because they would need to open the sterile container for the tube on Mars.

Sample tube photo from (Perseverance Sample Tube 266

These are the detailed contamination requirements:

(Mars 2020: mission, science objectives and build)

Though as we saw they think they overachieved at 8.1 ppb of total organics and 0.7 ppb maximum for any tier 1 compound. They don't say which is most abundant but this would mean e.g. they could have 0.7 ppb of glycine and also 0.7 ppb of alanine because they are measured separately. It's not a total for all amino acids. They don't measure all the amino acids but just two representative ones to get an idea of the level of contamination.

By definition an ultramicrobacteria has a volume of at most 0.1 cubic microns (Size Matters: Ultra-small and Filterable Microorganisms in the Environment). A micron is a millionth of a meter or a 10,000 th of a centimeter. So a cubic micron has a volume of a trillionth of a cubic centimeter, or a mass of a trillionth of a gram, assuming density similar to water. So based on ultramicrobacteria at a tenth of a cubic micron each, there are 10,000 of them for every part per billion.

So those 120+ nanograms are equivalent to 1.2 million ultramicrobacteria per sample tube,. You'd find it hard to notice a few tens of thousands of them especially if they were related to terrestrial life - or based on some exotic biology that our tests don't pick up at all.

Spores would be a bit larger. The Martian conditions might favour spore forming microbes and we might find spores in the Martian dust. The b. subtilis spore, one of the smallest spore forming microbes, is typically 1.2 μm long and 0.8 μm wide so its volume is about a cubic micron, increasing to 1.8 μm long and 1.2 μm wide on hydration (Morphogenesis of Bacillus spore surfaces) . So there would be a thousand of those in each nanogram.

The "witness tubes" which collect terrestrial contamination in a dummy run of sample collection can help geologists subtract contamination to get a good idea of the original levels of organics
- but are not enough to reveal biosignatures when terrestrial contamination is likely orders of magnitude higher than biosignatures astrobiologists are interested in

The "witness tubes" which collect terrestrial contamination in a dummy run of sample collection can help geologists subtract contamination to get a good idea of the original levels of organics - but are not enough to reveal the minute traces of biosignatures astrobiologists are interested in

They use witness tube assemblies to detect terrestrial organics. They are cleaner than the normal sample tubes, cleaned to a very high level and sealed and only opened on Mars, and they have materials inside which detect any organics. They go through similar processes to the normal sample tubes - and then they do a dummy run where the witness tubes go through all the same handling procedures as a normal sample tube except they don't collect any sample. Instead they collect any contamination from the sampling apparatus. (The Sampling and Caching Subsystem (SCS) for the scientific exploration of Jezero crater by the Mars 2020 Perseverance rover : 30-31, )

As another control they have a "drillable blank assembly". This is a sterile silicon dioxide brick covered in aluminium. Perseverance drills a sample from it just as it would from the Mars surface and collects it in a sample tube in the same way as the other samples. They then know any organics in this dummy sample were introduced by the drilling and sample collection process (SCS) for the scientific exploration of Jezero crater by the Mars 2020 Perseverance rover : 5.). They also have a witness tube to witness the contamination of the drill bits themselves, from assembly through to launch and landing on Mars.

The idea is that they can subtract the organics they find in these witness materials from the organics in the contaminated samples to get an idea of what the samples were like before contamination. This will be very useful for geologists.

The problem for astrobiology - at least for the Perseverance samples - is that you are looking for very minute traces of biosignatures and 150 ng  of terrestrial contamination or even 8.1*15  = 121.5 nanograms is a lot of contamination. That  means that it is going to overwhelm the tiny traces you expect of past or present day life on Mars unless it is very abundant.

They expect to return some samples with over 100 ppb of organics, which would be 1,500 nanograms in a 15 gram sample. Compared to that, 150 nanograms or less, maybe only 121 nanograms seems little to a geologist.

However, astrobiologists are interested in biosignatures and recognizable organics. With even 70 million years of surface ionizing radiation those 1,500 nanograms are reduced to 1.5 nanograms of recognizable organics (Pavlov et al., 2022 . Rapid Radiolytic Degradation of Amino Acids in the Martian Shallow Subsurface: Implications for the Search for Extinct Life : 1100 – 1101) . The rest would be short carbon chains like oxalate, formate or it might have benzene rings as benzoate. So how you are going to spot those interesting organics amongst 150 nanograms of contamination? Curiosity's youngest age is 80 million years (Farley et al., 2014, In situ radiometric and exposure age dating of the Martian surface)and Perseverance can't measure exposure ages.

Perseverance could help here by collecting a sample from a recently exposed crater. They could get it down to less than 100,000 years of exposure age. In that case they might have 100  ppb of interesting organics.

However, even if we find a deposit as rich in past organics as our oil shales, then the most interesting organics, the original least altered organics, may be in small quantities and still modified after deposition through diagenesis

Diagenesis is a process of compaction under mild conditions of temperature and pressure. When organic aquatic sediments (proteins, lipids, carbohydrates) are deposited, they are very saturated with water and rich in minerals. Through chemical reaction, compaction, and microbial action during burial, water is forced out and proteins and carbohydrates break down to form new structures that comprise a waxy material known as “kerogen” and a black tar like substance called “bitumen”. All of this occurs within the first several hundred meters of burial.
(Oil shale)

Then, most likely it's mixed with infall from space, and is chemically altered and most of the original past life organics, if any, has been washed out or modified. So they are interested in very small trace amounts that might perhaps still be unaltered from 3 billion years ago, so even then the 8.1 ppb of contamination would likely overwhelm the organics they are interested in. If there are any biosignatures left they might be in small quantities - and especially so if Mars hadn't yet developed photosynthesis 3 billion years ago.

I cover that (below) See:

• Visually identical rocks in the same strata are usually interchangeable for geology - this is far from the case for astrobiology - we may need to search many rocks that are geologically identical to find samples of direct interest for past life

It's the same for present day surface processes. Astrobiologists are interested in small trace amounts of organics in the atmosphere and in the soil. So even then it will be hard to do much meaningful astrobiology with this level of contamination.

The terrestrial organics would overwhelm the trace organics they are interested in, unless we are lucky enough to return recognizable past microfossils (with a high chance whatever we find is controversial as for ALH84001), or large amounts of present day life.

But we can rescue this
- with bonus samples in CLEAN containers sent on the EIS for astrobiology
- a thimbleful of dirt, a few grams of dust, clean atmospheric samples, and a representative pebble from a recently excavated crater [IN NEPA BOTH]

Video: Open letter to NASA - how we can rescue NASA's mission plans with bonus samples in clean containers

The priorities of astrobiologists are different from geologists.

Present day life: with no in situ life detection, we'd likely need to drill into lots of boulders before we find patches of present day life if any.

For past life then we need to find rocks with a young exposure age.

This is why I suggested NASA and ESA collect bonus samples in STERILE containers sent on the ESA fetch lander (Walker, 2022, Comment posted December 20th). [I called it an ESA fetch rover but the plan now is a lander assisted by two Marscopters News Briefing: NASA's Perseverance Mars Rover Investigates Geologically Rich Area (Sept. 15, 2022 : 1: 02 : 00) also briefly at (31:44)]

The aim of these bonus samples is to return

• A thimbleful of dirt (the more the better of course)
• Salts either in the same sample or ideally a second sample if there is a nearby salt concentration
• A litre of compressed atmosphere (using a similar compressor to MOXIE)
• A few grams of dust caught in the filters for the atmospheric compressor
• A pebble from a recently excavated crater - based on cratering statistics for Mars there should be a crater within reach only a few tens of thousands of years old excavated to a depth of 2 meters or more

Pavlov et al suggested looking for recent impact craters and rocks exposed by rapid wind erosion or perhaps in a deep valley.
(Pavlov et al., 2022, Rapid Radiolytic Degradation of Amino Acids in the Martian Shallow Subsurface: Implications for the Search for Extinct Life : 1113)

For that last comment on returning samples from a young crater, I have a rough calculation in the preprint:

• Cratering rate for 16 to 32 meter diameter craters of 2.57 craters per square kilometer every ten million years [ (1.9 + 0.67 for first two rows in table 1 of (Utilizing decameter scale crater populations to study Martian history]
• An observational study by Daubar et al. found that this size of crater excavated the surface to depths of between 2 and 9 meters [based on seven newly formed craters at this size in (The morphology of small fresh craters on Mars and the Moon. : Figure 4).]
• Based on those figures, 2616 craters of this size formed in the last 10 million years in any radius of 18 km,
• Perseverance can travel 18 km in 90 days at 200 meters a day if it was a priority
• Which leads to a 99.9999999996% chance of a crater less than 100,000 years old within 90 days travel of Perseverance

This graphic shows the results of the calculation (not yet peer reviewed)

Details in my preprint: (Walker, 2023, NASA must protect Earth's biosphere even if Mars samples hold mirror life): With yet more ambition we can search for past organics using a Marscopter to return pebbles excavated by a recent small impact crater to a depth of 2 meters or more with near certainty of exposure age less than 30,000 years)
(not yet peer reviewed, illustrative rough calculation)

A crater excavated to 2 meters may not be deep enough

Specifically, in a rock at 2 m depth and the exposure age of 500 million years, the original (primordial) abundance of amino acids would be decreased by a factor [a thousand] if a rock is a silicate and perchlorate-free and by a factor of [100,000] if a silicate rock at that depth contained 1% [of] perchlorate.

(Pavlov et al., 2022, Rapid Radiolytic Degradation of Amino Acids in the Martian Shallow Subsurface: Implications for the Search for Extinct Life : 1111)

However, this size of crater, 16 to 32 meters in diameter excavates to a depth from 2 meters up to 9 meters. There seems a reasonable chance of a crater excavated to many meters with an exposure age less than 100,000 years.

It's more tricky to return a sample from a young crater when we only have the Marscopters for the Europa lander. However if the Marscopters are able to communicate at a distant enough range to reach the edge of the landing ellipse for the ESA fetch lander, it may be possible to land it within range of a young crater, and then send the marscopters on multi-day journeys to travel to the crater - after a preliminary reconnaissance of the route by Perseverance which could also return a sample of organics from the crater.

This would seem to be a high priority target for astrobiology, to find such a crater and send Perseverance to investigate it.

This shows how the atmospheric compressor works. It’s using a proposal submitted to the decadal survey in 2020 by Jakosky et al.

First it uses the getter to remove evolved gases from the container wall. Then it closes one microvalve and opens another to get an atmospheric sample. Finally it closes both microvalves to the gas container and opens the vent to run more atmosphere through the compressor to collect dust in the filter

(Scientific value of returning an atmospheric sample from Mars)

We can detect life from the Gobi and Taklimakan Deserts in dust in Japan (Aeolian dispersal of bacteria associated with desert dust and anthropogenic particles over continental and oceanic surfaces).

Text on graphic: Microbes in dust from the Gobi desert detected in Japan (Yonago).

Martian dust storms might transport dead or alive microbes great distances.

Graphic: distance Gobi desert to Yonago

So we may have a chance of detecting viable or recently dead life from far away on Mars brought to Jezero crater in the dust storms.

For this we need:

• A sterile scoop for the ESA fetch rover
• Sterile containers and the atmosphere compressor also sent with the fetch rover
• A way for a Marscopter to pick up a pebble from a newly excavated crater without contaminating it and drop it in front of the ESA fetch rover so that it can pick it up in the scoop.
  [or the ESA fetch rover visits the crater if it is accessible]

The pebble would ideally be from a rock that has organics in it, perhaps Perseverance could analyse the same stratum - but far from the pebble so as not to contaminate it.

It would help us to understand past organics better if Perseverance collects a sample of organics from a recently excavated crater. There might be enough of the undegraded organics to not be overwhelmed by the forwards terrestrial contamination of the Perseverance samples.

However we saw that the organics in Mars meteorites are not necessarily the levels of organics we'd be looking for, for past life searches. Much of it may be produced natively on Mars in an abiotic fashion, infall from space or altered. We need to collect a bonus sample from the most interesting geological layer they found, in a clean container for useful astrobiology looking at the very minute traces they are interested in.

We need to take great care to protect Earth's biosphere with bonus samples too
- Mars could have astrobiological surprises as astonishing as the carbon dioxide geysers were for geology
- like mirror life [IN NEPA BOTH]

Video: Open-letter to NASA - Illustrative worst-case scenarios of mirror life and new or alien fungal genus

However we have to take great care with these bonus samples too.

NASA has made many extraordinary geological discoveries on Mars, such as the CO2 geysers.

Text on graphic: Artist’s impression of CO2 geysers on Mars, one of many geological surprises.

Mars could have astrobiological surprises too.

What if Mars has independently evolved mirror life?

Artist’s impression by Ron Miller of the martial CO₂ geysers that form in spring in the polar regions (PIA08660: Sand-Laden Jets (Artist's Concept), JPL).

So, why not something as surprising as life based on mirror chemistry or even more exotic.

As professor Sara Walker put it:

no one expects a geological map from Earth to exactly apply to Mars (or any other planet), yet there is widespread expectation a map of features of life on Earth will apply, I do not understand the logic

— Sara Walker (@Sara_Imari) November 8, 2021

I was using Mars as a specific example of a general phenomenon, but I do agree in the specific case of Mars it could be very similar (with the big assumption life is on Mars) if it has common origin with Earth, but not if independent

— Sara Walker (@Sara_Imari) November 8, 2021

Illustrative worst case scenario for effects on Earth's biosphere: INDEPENDENTLY EVOLVED MIRROR LIFE: according to one theory - punctuated chirality - there's a 50 - 50 chance that independently evolved life on Mars arose from a network of mirror organics interacting with each other - and still uses mirror organics

When a molecule can occur in two mirror forms, like your hands, it’s called chiral - the word chiral is derived from the Greek word χειρ (kheir) for hand. Terrestrial life is homochiral, which means that nearly all of its asymmetrical (chiral) molecules occur in only one of its two mirror forms. Also terrestrial life for the most part can’t use any mirror organics it finds and just ignores them.

According to one modern theory - punctuated chirality - there’s a 50 - 50 chance that independently evolved life on Mars uses mirror organics.

According to this theory, early on as life was just starting to evolve, there were patches of chemicals that worked together with each other in chiral networks which expand converting a non chiral substrate into chiral organics and where two chiral networks of opposite chirality meet there are ways for them to slowly convert each other to the opposite chirality.

There would be many such patches, some with the same chirality as terrestrial life and some with mirror organics. According to this theory, these patches would expand and flip each other back and forth in chirality on an environmental scale, with the chirality reset multiple times in Early Earth even if it didn’t go extinct (Punctuated chirality : 6) until one of them got established as the basis for the evolution of life.

If so, depending on how the flips went on Mars, life could easily have evolved from the mirror chemicals of the ones Earth life evolved from.

“Our analysis predicts that other planetary platforms in this solar system and elsewhere could have developed an opposite chiral bias.”

(Punctuated chirality : 7)

Text on graphic: By the theory of punctuated chirality, in a large sample of glucose from many planets containing life or prebiotic chemistry, roughly half would be D-glucose and half L-glucose

D-Glucose which terrestrial life can use

L-Glucose used as an artificial sweetener

Similarly for all organics that can occur in two mirror forms

Graphic for L-glucose and D-glucose by reflecting the graphic horizontally. Grey: Carbon, Red: oxygen, white: Hydrogen.

Detailed illustrative mirror life scenario
- an analogue of the blue-green algae chroococcidiopsis
- one of our best candidates for a Mars analogue organism
- can find all its nutritional requirements in basalt or gabbro, also sea water, fresh water and soil with strains found almost everywhere on Earth
- and a mirror life analogue on Mars would likely be adapted to metabolize normal organics because of infall from space [IN NEPA BOTH]

So, what if what we find on Mars is mirror life, or something even more exotic we haven’t considered before? It might be as remarkable as the CO2 geysers, but not safe to mix with our biosphere.

So that leads to this new scenario:

• Scenario of independently evolved mirror life, evolved from the mirror chemicals on Mars. If Mars has independently evolved life, this could be mirror life (Punctuated chirality : 7) and if we return mirror life from Mars the worst case could be quite dire, mirror organics spreading through our soils, of no nutritional value to our microbes, grass etc. Meanwhile in this scenario, mirror life can probably use terrestrial organics, accustomed to infall of both types of organics from space on Mars (Mirror-image cells could transform science-or kill us all)
   
• In this scenario, the mirror life is likely to be adapted to use normal organics because of infall from space, but few terrestrial microbes have the enzymes to convert mirror to normal organics to metabolize it
  
  Most terrestrial life depends on L-amino acids and can't use D-amino acids. However, the strain Raineyella antarctica LZ-22^T is able to use both L and D forms of the amino acids Met (methionine) and Leu (Leucine, and is only able to use the D forms and not the L forms of His (Histidine), and Val (Valine) (Amino acids and their abbreviations). See: (Pikuta et al.., 2016. Raineyella antarctica gen. nov., sp. nov., a psychrotolerant, d-amino-acid-utilizing anaerobe isolated from two geographic locations of the Southern Hemisphere) summarized in (Aliashkevich et al.,., 2018. New insights into the mechanisms and biological roles of D-amino acids in complex eco-systems : D-Amino Acids as a Sole Carbon and Nitrogen Source)
   
• We won’t want to find out what happens if mirror life spreads through our planet converting normal to mirror organics that only mirror life can use.
   
• Terrestrial life might well still eat mirror organics, like artificial sweeteners, but starve in the midst of plenty because it couldn’t do anything with the flipped starches or proteins, and many fats would also be inaccessible.
  
  For some of the differences see: (An adventure in stereochemistry: Alice in mirror image land) answers in: (Through the Looking Glass and What Alice Ate There). It’s quite tricky telling the difference between a molecule that’s chiral (different from its mirror image) and achiral (same as its mirror image) molecule - if you can just turn it over to get the other pattern it’s achiral, but if you have to reflect it in 3D as in a mirror it’s chiral. A carbon atom with four distinct groups attached to it is normally going to be chiral and life can usually only use one of the two forms - see (Chiral Molecules)

Text on graphic: Normal life, Mirror life, DNA, amino acids, sugars, fats, everything flipped. Most normal life can’t eat mirror organics. Martian mirror life might be able to eat normal organics.

To be more specific, in this scenario we might have a mirror life analogue of chroococcidiopsis on Mars.

• Chroococcidiopsis, a highly resilient blue-green algae does well in Mars simulation chambers (… Chroococcidiopsis sp. Exposed To a Mars-Like Near Space Environment) and is able to repair extensive DNA damage including double strand breaks, taking about 12 hours to repair the damage, with no significant loss of viability even after revival after the equivalent of 20 million years of surface ionizing radiation on Mars (Role of DNA repair pathways in the recovery of a dried, radioresistant cyanobacterium exposed to high-LET radiation: implications for the habitability of Mars : 9 - 10)
   
• It can find all its nutritional requirements in seawater, or rocks such as basalt, so long as it also has access to water and a source of energy such as sunlight or hydrogen. It's found even 10 to 750 meters below the Atlantic sea bed using an alternative metabolic pathway to metabolize hydrogen and with nothing else except gabbro to live on (Recycling and metabolic flexibility dictate life in the lower oceanic crust : 3). It is able to live there in small populations by using carbon and nitrogen found in small quantities in the basalt - though it can also use photosynthesis to add to the carbon when it has access to sunlight and can do nitrogen fixation to add to the nitrogen when it has access to nitrogen.
  
• A single monolayer of grains 0.15 mm thick reduces the UV nearly 100-fold (Absorption and Scattering of UV and Visible Light Through Simulated Martian Regoliths and Rock Samples : 5) and (from abstract) iron oxides in the Mars dirt preferentially block out damaging UV and lets through frequencies of light used by photosynthesis - a microbe protected by even a single grain of JSC Mars 1 simulant has orders of magnitude less UV damage. In that 2023 paper, Golin et al. concluded: "From the perspective of the radiation environment, a radioresistant microorganism that uses photosynthesis as an energy source could survive in the subsurface of Mars." (Absorption and Scattering of UV and Visible Light Through Simulated Martian Regoliths and Rock Samples : 10).
   
• Many minerals found on Mars similarly let light through and block damaging UV, like calcite, and kieserite a magnesium sulphate mineral abundant on Mars (The spectral variability of kieserite (MgSO4·H2O) with temperature and grain size and its application to the Martian surface
  , 2014). The best of all in this respect is the clay mineral cheto bentonite. These all block out damaging UV much more than they block the light needed for photosynthesis (Absorption and Scattering of UV and Visible Light Through Simulated Martian Regoliths and Rock Samples : 9)
  

• This blue-green algae could live anywhere on Mars with basalt and access to water.
   
• Chroococcidiopsis is an ancient polyextremophile with numerous alternative metabolic pathways that strains of this genus can use, including nitrogen fixation, methanotrophy, sulfate reduction, nitrate reduction etc (Metabolic pathways – Chroococcidiopsis thermalis). An alien microbe from Mars based on mirror life might be just as versatile, accumulating many pathways over billions of years of evolution on Mars. That would help it to radiate through our biosphere even if we return only one species from Mars.

Not Chroococcidiopsis flipped in a mirror, that would be absurd. But an independently evolved mirror life analogue of our blue-green algae on Mars with similar nutritional requirements and environmental preferences, but made up of mirror organics.

Some synthetic biologists have embarked on a likely decades-long research project to make mirror life in the laboratory by slowly flipping components of a terrestrial microbe bit by bit.

Synthetic biologists will ensure mirror life is not able to survive in the wild at any point in this slow step by step transformation from normal to mirror life. In Kasting and Church’s worst-case scenario for release of mirror life from a terrestrial lab, mirror life retains the edge over normal life in this evolutionary race and eventually there is little left except mirror organics and life that can use it. They suggest humans could go extinct in this worst case scenario that they need to avoid, at least in natural ecosystems

“—both Kasting and Church think mirror predators would evolve, but whatever life existed on Earth by that point wouldn’t include us.”

(Mirror-image cells could transform science-or kill us all)

It would be a similar scenario if we return naturally evolved mirror life from another planet.

I don’t think we’d go extinct in that scenario. We might be able to engineer terrestrial microbes with the ability to eat mirror organics and limit the mirror life, and in the worst case we could “paraterraform Earth” - we could cover jungles, grassland, mountains, islands, coral reefs, eventually continents, ice sheets, and large areas of the oceans with larger and larger shelters like Biosphere 2 (Under the Glass Systems) and with the technology of the future, manage them internally to keep out most of the mirror life. But if we do have mirror life on Mars, we as a civilization would surely decide not to do the experiment of returning it to Earth.

The mirror life could also co-exist with normal life could have evolved in regions separated by physical barriers, for instance after the volcanic eruption on the flanks of Arsia Mons 210 million years ago, which likely lead to 100 cubic kilometers of subsurface melt in three lakes, which would have stayed melted for centuries to millennia insulated by surface ice (A habitable environment on Martin volcano?)

With the punctuated chirality model, Gleiser et al. see it as an unlikely possibility that the very early stage of prebiotic chemistry they are looking at could start in hydrothermal vents, but if it could, different vents would vary in chirality. However, there is too much mixing from the circulation of the oceans for them to stay as separate networks indefinitely, as they gradually evolve to life (Punctuated chirality : 6).

Mars would have many more opportunities for separate networks early on with less water in its ocean, and separated impact basins. Perhaps that might suggest it could have co-existing normal and mirror life even early on.

This is a combination of two scenarios in Cockell's paper on trajectories of habitability on Mars, the idea of a new biologically isolated habitat that forms by impact into permafrost::

A scenario for the formation of an uninhabited habitat on Mars by an impact into permafrost, which remains hydrologically isolated at macroscopic and microscopic scales even on a planet that has a hypothetically colonized subsurface
( Cockell, 2012, Trajectories of Martian habitability : : fig. 6)

Which is combined with the idea of life becoming extinct and reoriginating on Mars - in this case it only becomes locally extinct and is still present elsewhere on Mars.

There are other trajectories of greater complexity that can be envisaged. Examples include an inhabited Mars on which life becomes extinct and then reoriginates (or is transferred from Earth) at some later time.

( Cockell, 2012, Trajectories of Martian habitability)

If the habitat forms as a result of a volcanic eruption then it could remain habitable, but isolated beneath a surface layer of ice, perhaps for some considerable period of time. Also if life never developed photosynthesis or this happened before photosynthesis, uninhabited habitats on the surface could become biologically isolated more easily. Similarly if it never developed resilient spores or other propagules able to spread in the dust storms any life would likely be very localized.

Whether this is possible would depend on how long it takes for a new form of life to evolve, and how much time the habitats persisted for at various times in Martian history and the capabilities of any life that does evolve.

It might be possible for very early life to co-exist with terrestrial life if its cells are very small, based on mirror life or some other biology. That was the inspiration behind the hypothesis of a shadow biosphere of nanobes ( Cleland, 2019, The Quest for a Universal Theory of Life: Searching for Life as we don't know it : 213 - 214) which could co-exist with modern life. Earth doesn’t seem to have a shadow biosphere (yet) but small cells have an advantage in an environment with low nutrient concentrations (Ghuneim et al., 2018 ( Nano-sized and filterable bacteria and archaea: biodiversity and function.)

• very small cells can escape grazing by larger grazing amoebas which don’t notice them
• very small cells have a larger surface area to volume ratio, so can use nutrients better in nutrient poor conditions, as they have a larger surface to volume ratio, and so take up nutrients more efficiently. They would also avoid protozoan grazing 

If ribocells are possible they could be very tiny (SSB, 1997, Size limits of very small microorganisms: proceedings of a workshop : 117). . These are cells that have only RNA, no DNA, no proteins, and the enzymes are made up of fragments of RNA. It is a field of very active research at present (Kun ,2021. Maintenance of Genetic Information in the First Ribocell). If at some point in our exploration of Mars, we find very small early life cells, it doesn't seem that we can assume in advance that they couldn't survive alongside more evolved terrestrial life. We would need to evaluate them carefully to see if they can be safely returned to Earth unsterilized.

We do have a potential analogue of this happening in the past. We don't actually know that photosynthetic life originated on Earth. If it got to Earth from Mars, a minority view, it could have been the largest ever transformation of Earth's biosphere the result of potentially just one viable microbe transferred from Mars to Earth. Life could have originated on Mars and it might by chance have evolved photosynthesis first there, perhaps because conditions on Mars for some reason favoured the evolutionary pathways leading to photosynthesis.

• The Space Studies Board give no particular example of a way that a novel species from another planet could cause a mass extinction
  - however the Great Oxygenation Event is a good illustration of how a single microbe transferred between planets could cause major changes to another planet's biosphere and
  - It's not known if the Great Oxygenation Event caused a mass extinction or not
  - but it is an example to show that a single species can in principle cause major changes to a biosphere as with our scenario of mirror life
  - with potential to cause mass extinctions as a result

Illustrative worst case scenario for human health and wildlife (especially birds):
- NOVEL GENUS OF FUNGI from Mars able to infect us similarly to Aspergillus fumigatus
- harms 200,000 immunocompromised a year, with high fatality rate 30% to 95%
- harms 48 million with an allergic lung disease (ABPA, 2.5% of asthma cases)
- harms 400,000 with a serious chronic allergic lung disease
- and not adapted to an infectious lifestyle in any organism
[NEW detailed scenario, fungal pathogens IN NEPA BOTH]

I also developed a plausible scenario for a serious human pathogen to motivate taking great care to protect human health from large-scale impacts. It is also a good example to motivate taking great care with the Mars samples to protect birds (wild or domestic).

This particular example of a novel fungal genus similar to Aspergillus is new, but I cover fungal diseases from Mars in the preprint that I was working on before the first round of public comments. The version I attached to the second round of public comments is here:

• NASA and ESA are likely to be legally required to sterilize Mars samples to protect the environment until proven safe

I'd like to emphasize again for anyone who gets easily scared - this is a worst case scenario like a house fire. Not intending to suggest in any way that this is a highly likely scenario for a fungus from Mars.

So here is the scenario:

• a fungal disease, similar to Aspergillus, which is not adapted to humans and has severe effects on 200,000 immunocompromised people a year, fatality rate 30% to 95% (Hidden killers: human fungal infections : table 1), and in immunocompetent people, it causes allergic reactions in 2.5% of all asthma cases. That’s typically 48 million cases of ABPA (allergic bronchopulmonary aspergillosis), which in 400,000 cases have progressed to the serious chronic disease of chronic pulmonary aspergillosis (Global burden of allergic bronchopulmonary aspergillosis with asthma and its complication chronic pulmonary aspergillosis in adults) for details of the disease: (Allergic Bronchopulmonary Aspergillosis (ABPA) Symptoms, Diagnosis, Treatment & Management)
  
• Aspergillus fumigatus (the main Aspergillus species to harm humans) has no known specific adaptations to infect any other organism, human or otherwise (Aspergillus fumigatus: contours of an opportunistic human pathogen). Instead it infects us because of many adaptations to extreme environments (Ecology of aspergillosis: insights into the pathogenic potency of Aspergillus fumigatus and some other Aspergillus species)
   
• with characteristics that may well be shared by martian fungi (Ecology of aspergillosis: insights into the pathogenic potency of Aspergillus fumigatus and some other Aspergillus species : table 1, table 2 and section: Biophysical capabilities and ecophysiology of pathogenic Aspergillus species).

Paulussen et al put it like this:

Collectively, the aspergilli are remarkable fungi. … there are numerous aspects of Aspergillus cell biology and ecology (including their metabolic dexterity when adapting to nutritional and biophysical challenges) which contribute to their status as, arguably, the most potent opportunistic fungal pathogens of mammalian hosts.

Aspergillus species are able to utilize a wide range of substrates, highly efficient at acquiring such resources, and can store considerable quantities of nutrients within the cell; all traits which contribute to their energy‐generating capacity and competitive ability

…

Species of Aspergillus are also among the most stress‐tolerant microbes thus far characterized in relation to, for example, low water activity, osmotic stress, resistance to extreme temperatures, longevity, chaotropicity, hydrophobicity and oxidative stress

(Ecology of aspergillosis: insights into the pathogenic potency of Aspergillus fumigatus and some other Aspergillus species : section: Biophysical capabilities and ecophysiology of pathogenic Aspergillus species).

Those are all capabilities that would be very useful on Mars in the high stress, highly oxidising, low nutrient conditions there, as well as other adaptations of Aspergillus mentioned in that paper such as ability to respond to rapid hydration and ability to cope with low oxygen conditions in the lungs.

Also Aspergillus can form aerosols very easily - spores floating in the wind - and a fungus on Mars might do the same, to propagate in the very thin atmosphere on Mars. For a rough idea, typical winds are roughly as fast on Mars as Earth or faster, over 45 meters per second reaching over 160 kilometers per hour in dust devils (HiRISE Clocks Hurricane Speed Winds In Martian Dust Devils), but at typically less than a hundredth of the density, that's equivalent to 16 kilometers per hour (since kinetic energy is mass * velocity squared) or about Beaufort scale 3, which is not quite enough to lift dust and loose paper (Beaufort scale). Even with the low gravity the only reason that Mars has dust storms is because the particles of dust are very small, up to a few microns in diameter.

This shows how aspergillus fumigatus infects the lungs. First the wild fungus produces spores. The spores settle in the lungs. They then penetrate the lungs with hyphae (tendrils) which extract organics. It protects itself with chemical barriers and produces branching networks of these tendrils which then break up and spread through the body.

Text on graphic. The way a fungus infects us is very straightforward - insert tendrils (hyphae) to extract organics. Even an alien fungus with no adaptations to terrestrial life could do this. Uses chemicals to attack tissues. Also protects itself with chemical barriers.

Graphic from: (Aspergillosis - Creative Biolabs)

We already have a Mars analogue black yeast Exophilia Jeanselmei which can grow in Mars simulation chambers and is classified as a risk group 2 organism in humans.

• The NRC study dates from 14 years ago and a new study would find examples of Mars analogue organisms that can be human pathogens such as the black yeast Exophiala jeanselmei MA 2853 which is found in rocks in Antarctica as well as in warmer conditions, and can grow up to human blood temperature, risk group 2 (moderate individual risk, low community risk)

The main thing that may be different from a Mars organism is that Aspergillus fumigatus needs oxygen, but it can manage with levels of oxygen as low as 1% at sites of infection where the tissues take up oxygen to fight back, while the related Aspergillus Terrus can manage in lungs without any oxygen using nitrogen reduction (Ecology of aspergillosis: insights into the pathogenic potency of Aspergillus fumigatus and some other Aspergillus species : table 2). Many fungi don't need oxygen, also Mars has some possibility of oxygen in colder Martian brines (O2 solubility in Martian near-surface environments and implications for aerobic life, 2018). Also the fungal component of the alpine lichen Pleopsidium chlorophanum found on rocks from California to Antarctica seems to get all the oxygen it needs from the algal component growing in Mars simulation chambers.(Adaptation of an Antarctic lichen to Martian niche conditions can occur within 34 days). Perhaps a martina fungus could also grow in a biofilm that includes photosynthetic life. So we might well return fungi from Mars that are able to use oxygen as well as to grow without oxygen.

This is a close up of the details of how a spore (conida) lands on a surface and starts to grow into it.

Though the details are complex the basic way a fungus like aspergillus infect us is rather straightforward - it uses tendrils to extract organics and it protects itself with chemical barriers. It also uses chemicals such as acids or alkalis to damage the host tissue to help with breaking up the organics and to protect itself. These are things that even a fungus analogue from an alien unrelated biology might do.

Graphics from (Aspergillosis - Creative Biolabs).

A novel fungus like Aspergillus doubles as an illustrative worst case scenario for wild birds - birds are especially severely affected because they don't have an epiglottis, can't cough and have fewer cilia to remove spores from their lungs

This also doubles as a worst case for wildlife as Aspergillus is a serious pathogen of some wild birds (Aspergillosis in mammals and birds: impact on veterinary medicine) especially wildfowl, raptors and gulls (Cornell Wildlife Health Lab).

It also doubles as a worst case scenario for domesticated birds because Aspergillus often causes serious problems with turkeys and other poultry in farms. There is no treatment available for commercial flocks though pet birds can be treated (Aspergillosis in Poultry)

• Aspergillus is especially serious in birds, because they don’t have an epiglottis to prevent particles getting into the lower respiratory tract, or a diaphragm for a cough reflex, and have fewer cilia to move foreign bodies out of the lungs : (Aspergillosis in mammals and birds: impact on veterinary medicine).

Our body doesn't have broad spectrum antifungals like it has for bacteria and archaea because they have neutral cell walls like our own cells - so it uses patterns specific to each genus and might either ignore a novel genus from Mars or else overreact with an allergic reaction - and there may be no antifungals to treat it

The details here are technical and I go into them in more depth below:

• We are protected from Aspergillus by specific adaptations of our immune system to recognize this genus - without this fine tuned reaction our bodies might either ignore or over react to a novel fungal genus of either terrestrial or alien biology it has never encountered before - and there might be no antifungals available that work with it.

The mode of action of a fungal disease could also be used by an alien biology - because of its simplicity - inserting tendrils to extract organics from an organism

This is motivated by Joshua Lederberg's statements that

Whether a microorganism from Mars exists and could attack us is more conjectural. If so, it might be a zoonosis [infectious disease that jumps to humans] to beat all others.

Quote from: (Parasites face a perpetual dilemma)

Also by the discovery of dark matter fungi. Researchers found these dark matter fungi as a result of advances in rapid gene sequencing. They called them “Dark Matter Fungi”, so called because they are not easy to study or cultivate (Discovery of dark matter fungi in aquatic ecosystems demands a reappraisal of the phylogeny and ecology of zoosporic fungi). They found them in photoysnthetic life in freshwater and seawater including cyanobacteria and diatoms.

Text on graphic: How chytrid fungi attack diatoms. Alien organisms could do the same, insert filaments to extract organics.

Description: false-colour red shows chytrid-like [zoo-]sporangium structures.

(Chytrid fungi distribution and co-occurrence with diatoms correlate with sea ice melt in the Arctic Ocean - Communications Biology : Figure 7)

Early research focused on fungal diseases of fresh water microalgae (phytoplankton) such as diatoms and blue-green algae. However more recently, scientist discovered that there are many fungal diseases of marine microalgae too. They just found the freshwater ones first. A review from 2022 describes them in the title of the paper as a “mystery yet to unravel” (Basal parasitic fungi in marine food webs—a mystery yet to unravel))

I discuss this and various other analogues for alien life such as microplastics, sterile inflammation such as silicosis and misincoporation of amino acids leading to protein misfolding with BMAA in supplementary material below. See:

• The gnarly question of our vulnerability to a completely alien biology - “Whether a microorganism from Mars exists and could attack us is more conjectural. If so, it might be a zoonosis [infectious disease that jumps to humans] to beat all others” - Joshua Lederberg (discoverer of bacterial sex and pioneer in planetary protection)

These are illustrative worst cases
- there are many ways samples can be harmless
- no life
- not able to spread
- can be contained in a BSL-4 or
- first microbes from Mars are harmless [IN NEPA BOTH]

These are worst cases.

• We most likely don’t return life at all
• if we return life, it most likely wouldn’t be able to spread outside the laboratory,
• a BSL-4 might well be able to contain the life,
• the first microbes from Mars might well be harmless or even beneficial (most introduced species are on Earth)
• It’s especially unlikely our first samples returned from another planet returns something like either of these scenarios in a mission that isn’t able to detect present day life on Mars and given the harsh conditions on Mars.

In all the sample return reports to date, the experts have been of the view that the chance of returning a microbe capable of large-scale harm to human health or the environment like these examples appears to be low.

We may need vivid and clear illustrations like this to motivate space agencies like NASA to pay closer attention to how they develop their environmental impact statements

We need to prepare for worst case scenarios, and not just best case scenarios. We also need to set a good precedent for future samples returned from Mars.

In my literature survey, I found the existing planetary protection literature doesn’t have many actual concrete examples. However we may need vivid illustrations now, to help motivate space agencies to take more care. I have many more scenarios in the preprint. Some are of especial interest for alien life such as alien fungi, or alien life that doesn't respond to terrestrial life like microplastics or nanoplastics but could still harm us. See separate page: Main points in the open letter in more depth - selected highlights

NASA may be able to contain ultramicrobacteria and gene transfer agents in a biosafety laboratory with new technology - but this capability doesn't yet exist and needs to be developed first [IN NEPA BOTH]

You might be able to fulfill the ESF requirements with new technology. This technology would need to be developed, methods of testing it, repairing it, etc devised - which could take some years. The technology doesn’t exist yet as none of our biosafety laboratories need to contain ultramicrobacteria.

Even the most well prepared biosafety laboratory has to have emergency procedures for lab leaks

However, any biosafety laboratory with human technicians has to have emergency procedures in place for lab leaks.

Even the most well prepared laboratory may experience unintentional or intentional incidents or emergencies despite existing prevention or risk control measures.

(WHO, 2020, World Health Organization Laboratory biosafety manual, 4th edition : 87)

The Apollo missions had several lab leaks and their technicians went into quarantine
- but this was before NEPA
- the quarantine precautions were decided by NASA
- not an agency established to provide health expertise
- there was no peer review
- it was not open to public comment
- their precautions would not keep out mirror life or fungal diseases of crops or fungal diseases of humans
- or diseases with life long symptomless human spreaders (similarly to typhoid Mary)
[IN NEPA BOTH]

Video: Open letter to NASA - quarantine can't contain mirror life etc - telerobotics can - easiest in orbit

The Apollo missions used quarantine for technicians - Technicians for the lunar receiving laboratory had to go into quarantine at least twice after a breach of sample containment during sample handling, 11 technicians in an incident for Apollo 12 and 2 for Apollo 11 (Meltzer, 2012, When Biospheres Collide : 241. 485).

However this was before NEPA. The Apollo procedures were decided internally and only released on the day of launch. They were never subject to legal review or public scrutiny (Meltzer, 2012, When Biospheres Collide : 452).

Quarantine of technicians won’t work for mirror life, or fungal diseases of crops or humans, or diseases with lifelong symptomless human spreaders. which could set up home harmlessly in the human microbiome.

The Apollo procedures wouldn’t keep out a life-long symptomless spreader of a pathogen similar to Mary Mallon for typhoid (Mary Mallon: First Asymptomatic Carrier of Typhoid Fever).

We’ll also see quarantine can’t work for fungal diseases of crops, and there are many other examples where quarantine of technicians can’t protect Earth.

For a vivid illustration, the ISS has a preflight “Health stabilization program” which uses vaccination and a 14 day quarantine to help prevent upper respiratory infection and gastroenteritis (Health Stabilization Program V1 4.4.2.4) but of course this can’t keep out fungi that are harmless to young healthy astronauts. Two of the Zinnia plants died from a fungal pathogen probably brought there by one of the astronauts.

Text on graphic: Mold growing on a Zinnia plant in the ISS. The mold fusarium oxysporum likely got to the ISS in the microbiome of an astronaut (Draft genome sequences of two Fusarium oxysporum isolates cultured from infected Zinnia hybrida plants grown on the international space station). (How Mold on Space Station Flowers is Helping Get Us to Mars)

This fungal disease fusarium oxysporum is also an occasional opportunistic pathogen of humans (Genomic Characterization and Virulence Potential of Two Fusarium oxysporum Isolates Cultured from the International Space Station)

This is just intended as an illustration to show that it's impossible to use quarantine to keep out a fungal disease. In this case, the fungus didn't kill the plants by itself. First, the plants got too much water, then four got mold. After a change to a higher fan speed two of the four infected plants recovered but two of them died (How Mold on Space Station Flowers is Helping Get Us to Mars) The strains isolated from the ISS weren’t able to infect healthy Zinnia plants (Genomic Characterization and Virulence Potential of Two Fusarium oxysporum Isolates Cultured from the International Space Station : 13). Other strains of fusarium oxysporum were found in isolates from the dining table in the ISS. (Genomic Characterization and Virulence Potential of Two Fusarium oxysporum Isolates Cultured from the International Space Station). Also we can’t know how this fungus got to the ISS, but it could be brought there on an astronaut’s microbiome.

However it's a vivid example to show how we can't keep fungi out of the ISS and it would be the same for any Martian fungi that can live in or on humans, most likely we wouldn't be able to keep them inside a quarantine facility. There are many other fungi on the ISS including Aspergillus fumigatus (Characterization of Aspergillus fumigatus Isolates from Air and Surfaces of the International Space Station) which is not likely to cause problems for healthy young astronauts and is commonly found in buildings.

The Apollo precautions also couldn't work with pathogens with long latency periods. Carl Sagan used the example of Leprosy

Carl Sagan gave the example of leprosy for the “vexing question of the latency period”

There is also the vexing question of the latency period. If we expose terrestrial organisms to Martian pathogens, how long must we wait before we can be convinced that the pathogen-host relationship is understood? For example, the latency period for leprosy is more than a decade.

(Sagan, 1973, The Cosmic Connection – an Extraterrestrial Perspective : 130)

We now know that leprosy can take 20 years or more to show symptoms (WHO, 2019, Leprosy, Key facts,)

The draft EIS has nothing about quarantine
- based on NEPA statements for other BSL-4s, it says the risk to the public from a typical BSL-4 can be described as zero for direct release and is negligible even if workers get contaminated
- it has no mention of quarantine or precautions for lab leaks
- no suggestion that there is anything different about a Mars sample receiving facility and a normal BSL-4
- the big difference is that in a sample receiving facility we don't know what needs to be contained, what its capabilities are, or even if it is based on terrestrial biology while
- with a BSL-4 the experimenters know what they are studying

This is all the EIS says about the possibility of accidental release from the Mars sample handling facility. They say the risk can be described as zero for accidental release from the facility and is negligible in case of a lab leak.

It relies on submitted final Environmental Impact Statements for other biosafety level 4 laboratories. NASA's team don't provide any analysis of their own at this point. NASA's team concludes:

While not completely analogous, the results of previous NEPA analyses for BSL-4 facilities have concluded that the hazards associated with the operation of BSL-4 facilities are expected to be minimal.

Analyses performed in support of recent NEPA documents conclude that the risk from accidental release of material from a BSL-4 even under accident conditions that include the failure of protective boundaries (e.g., reduced effectiveness of ventilation filtration systems) are minute and can be described as zero (NIH/DHHS 2005).

An alternative release path resulting from the contamination of workers leading to direct contact with others (members of the public) was also analyzed [this refers to analyses in their two example BSL-4 EIS's, not an analysis by NASA]. Qualitative risk assessments for this mode of transmission [for the two previous EIS's for ordinary BSL-4 labs] have shown that the risk to the public is negligible (NIH/DHHS 2005, DHS 2008).

Should the Proposed Action be chosen, Tier II NEPA analyses of the proposed SRF11 would include analysis similar to those performed for existing BSL-4 facilities.

(NASA, 2023, MSR FINAL PEIS :3-14)

Cites:
NIH/DHHS. (2005). Final Environmental Impact Statement National Emerging Infectious Diseases Laboratories, Boston, Massachusetts. Bethesda, MD: National Institutes of Health, U.S. Department of Health and Human Services.
DHS. (2008). National Bio and Agro-Defense Facility Final Environmental Impact Statement. Washington D.C.: U.S. Department of Homeland Security

[My comments in red]

The argument here is that since the final PEIS of two other BSL-4s showed the risk to the public was negligible, they can assume that the risk to the public from a Mars sample return facility is negligible.

This of course is not a valid argument. The difference is that in a conventional BSL-4 the researchers know what they need to contain, and are not required to contain ultramicrobacteria, gene transfer agents and possibly mirror life, ribocells, and other forms of exotic biology. As we saw, the ESF in 2012 set requirements well beyond a BSL-4.

• The European Space Foundation in 2012 said we have to contain the far smaller ultramicrobacteria and gene transfer agents
  - 100% containment at 0.05 microns or larger and 1 in a million for release of a single particle ever at 0.01 microns or larger
  - well beyond capabilities of a BSL-4

And then there's the issue of how you respond to a lab leak when you have no information yet about any potential pathogens, and don't even know if any pathogen is based on terrestrial biology.

There is very little in the Environmental Impact Statement about the sample receiving facility - with most of the focus on sample retrieval and the capsule.

The only mention of the word "quarantine" in the document is a historical mention for Apollo and nothing about how they would respond to a lab leak leading to exposure of technicians to the samples.

Apollo quarantine was only designed to protect Earth from diseases with an incubation period less than 3 weeks
- and even for those, NASA's plan was to rush any technician or astronaut who got seriously ill to hospital as an authorized breach of quarantine

You might wonder - what about the Apollo era quarantine of astronauts and technicians? The Apollo quarantine wasn’t designed by NASA to keep out all infectious diseases of humans. Their aim was to try to ensure any infectious disease brought back from the Moon had a long enough quarantine period so that we would have time to put measures in place to slow down spread of the illness. Richard Bryan Erb was the manager of the Lunar Receiving laboratory from 1969 to 1970 (Carroll, 2019, The Early History of Canadian Planetary Exploration). He explained that the Apollo quarantine procedures were just intended to try to stop a pathogen with a short incubation period of less than 3 weeks

You never know whether something might show up in thirty years. There are viruses and things that will show up long after the fact, but the theory was that if you can go through a quarantine for three weeks, which was the time set, without adverse effect, then you're obviously not dealing with something that is rapidly reacting and dangerous, so you would have time to prepare a remedial action. It was a good trade, I think, between a hazard, which was not very likely, but a risk of perhaps life on Earth, which was immense.

(Butler, 1999, Edited Oral History Transcript).

However, if a technician or astronaut became seriously ill and needed urgent treatment that wasn’t available within the quarantine facility, NASA’s stated plan was to immediately take them out of quarantine and to a hospital:

If a serious medical emergency had occurred that was beyond the capabilities of CRA (Crew Reception Area) equipment, NASA would have rushed the afflicted person from LRL [Lunar Receiving Laboratory] to a hospital, regardless of quarantine requirements

(Meltzer, 2012, When Biospheres Collide : 229).

So even if there was a rapidly acting pathogen and all the technicians got seriously ill quickly, they’d have been rushed to hospital, in an authorized breach of quarantine, so it wouldn’t have worked that well as a way to protect human health. That then risks Hospital-acquired infections (nosocomial infections) which are often the main way an infectious disease spreads, for instance this is one of the main ways MERS spreads.

Nosocomial outbreaks have been a hallmark of MERS-CoV infections, and account for roughly a third of MERS-CoV cases reported globally.

(Hui et al., 2018, Middle East respiratory syndrome coronavirus: risk factors and determinants of primary, household, and nosocomial transmission)

In the Apollo era NASA had an interagency panel - but all discussions were private and it was set up so no changes could be made to NASA's plans without NASA's own approval

NASA had an interagency panel for the Apollo quarantine discussions, but the discussions were private, and it was set up so all parties had to agree on any changes to NASA’s plans, including NASA itself.

… the regulatory agencies agreed “not to take any actions that might have an effect on the lunar program … without the ‘unanimous recommendation of the agencies represented on the [Interagency] Committee [on Back Contamination].”

… Since NASA was itself a member of ICBC, no actions could be taken without its approval.

(Meltzer, 2012, When Biospheres Collide : 193).

NASA used those powers to tell the astronauts to open the Apollo 11 door while still floating in the sea - against the objections of the National Academy of Sciences whose representative Vishniac said it would make the rest of the quarantine program pointless

NASA used those powers to block recommendations by other agencies, They often did object. For instance this was the view of Vishniac of the National Academy of Sciences on the plans to open the Apollo 11 capsule door and exit into a dinghy in the open sea:

Opening and venting the spacecraft to Earth’s atmosphere after splashdown would, in his view, make the rest of Apollo’s elaborate quarantine program pointless.

(Meltzer, 2012, When Biospheres Collide : 452).

Carl Sagan saw it as part of NASA's (understandable) prioritization of the well being of their astronauts over planetary protection. He put it like this:

.The one clear lesson that emerged from our experience in attempting to isolate Apollo-returned lunar samples is that mission controllers are unwilling to risk the certain discomfort of an astronaut – never mind his death – against the remote possibility of a global pandemic.

... It was judged better to open the Apollo 11 hatch to the air of the Pacific Ocean and, for all we then knew, expose the Earth to lunar pathogens, than to risk three seasick astronauts.

(Sagan, 1973, The Cosmic Connection – an Extraterrestrial Perspective : 114)

But this was before NEPA. The Apollo procedures were decided internally and only released on the day of launch, and this was never subject to legal review or public scrutiny (Meltzer, 2012, When Biospheres Collide : 452).

So, the way NASA is behaving is very similar to the 1960s, NASA made decisions and other agencies had to follow their lead. This was easy back then, with almost no public awareness of the issues back then.

NASA is behaving in a similar way today - but the legal situation is now very different - as is public and general scientific opinion of the need to protect human health and the environment more rigorously

However, the legal situation is different now with NEPA. We also have far greater public interest and awareness in the potential for risks to Earth’s biosphere and human health. Though NASA could do this in the 1960s, they won’t be able to do it today, at least, not all the way through to 2033.

It was possible to design a mission even with 1960s technology to keep Earth 100% safe - and then with robotic technology and sterilized sample returns they could have quickly established that the Moon was uninhabitable

It is impossible to know what scientists and the general public would have decided in the 1960s, if NEPA had predated Apollo 11. However there were ways that we could have made the Apollo missions completely safe, for instance using robotic sample returns similarly to the Soviet missions. It wouldn’t have impacted on the science return much to sterilize the first robotic samples. Then send humans once the Moon was confirmed to be sterile.

This of course is not the situation with Mars, it's got an atmosphere, and water vapour close to the triple point, dust storms, we find liquid salty water on the surface, ice caps that vary seasonally, and so on, a very different situation. Which is why Sagan envisioned dozens of missions to explore Mars in situ first.

But in the case of the Moon, we could likely have sent astronauts to the Moon reasonably safely with only a delay of a few years using telerobotic exploration first, as for the Russian lunakhod missions, and sterilized sample returns until we could show that the surface of the Moon was uninhabitable.

Reminder to NASA
- you have a legal requirement under NEPA to consider substantive public comments such as the suggestion in my comments to use telerobotics to keep Earth 100% safe
- which is also a way to chart a route for the Titanic to find its way back to clear waters [NEW]

I'd like to remind NASA, of your:

• Legal requirements under NEPA to consider substantive public comments - and four NEPA requirements that your EIS doesn't seem to meet

In this open letter I suggest one possible direction to steer the NASA administration Titanic back into clear waters, avoiding the iceberg of public and scientific opinion in 2033, and maintaining your current world-leading role in planetary protection and Mars exploration science.

Because of quarantine issues
- unsterilized samples have to be returned to a telerobotic facility until we know what's there
- but likely costs well over half a billion dollars
- and many issues with a ground based facility
- including end of life sterilization for mirror life
- nobody has done this before
- and it also has to take account of accidents and criminal damage (for high levels of assurance)

I concluded because of quarantine issues, unsterilized Mars samples have to be returned to a telerobotic facility, until we know what’s in them.

We do have a design for a fully telerobotic Mars Receiving Facility, one of three designs submitted to NASA in 2009. The LAS study relies on telerobots to do almost all the sample handling. (Beaty et al., 2009, Planning considerations for a Mars sample receiving facility: Summary and interpretation of three design studies :75). So we could do it already in 2009, and telerobotic capabilities have improved since then.

 

Sketch of telerobotic facility Credit NASA / LAS ( Keeping Mars Contained,)

The 2010 decadal review estimated the cost of the Mars sample receiving facility as $471 million in 2015 dollars (Mattingly , 2010, Mission Concept Study, Planetary Science Decadal Survey, MSR Orbiter Mission (Including Mars Returned Sample Handling)) or $659 million dollars today. Others estimate over half a billion dollars in 2015 dollars (The Plan to Bring Mars Down to Earth).

$471 milion in 2015 dollars equates to a little over $600 million dolars today.

That was using the 1999 size limit, essentially a BSL-4 but with the challenge that the samples need to be kept free of terrestrial contamination requiring a clean room inside a BSL-4, a BSL-4 inside a clean room or a novel double wall construction, all of which are new technology (Carrier et al., 2019, Science-Driven Contamination Control Issues Associated with the Receiving and Initial Processing of the MSR Samples : Figure 4).

Adding the ESF requirement would likely increase the cost considerably since it wouldn't be possible to use HEPA filters and some other approach would be needed. It would also extend the timeline since we first need to develop the technology to contain ultramicrobacteria and devise ways of testing the filters or air incinerator or whatever we use, repair it, replace it, all without letting a single particle of 0.05 microns or larger escape the facility.

At this level of funding, NASA will need to commit to Congress that the cost and schedule is adequate ( System Engineering Handbook : section 3.5). However they can’t do this until they know what to build and they won’t know what to build until they know the legal requirements which would require a new EIS. Another issue is that by NASA regulations, the build can't start until the technology is decided. It might be necessary to do sample return review and the updated size limit and level of assurance first. Then there is the time to build it, and train operators.

However it must be able to contain life to better than BSL-4 all the way down to gene transfer agents and ultramicrobacteria. The ESF required 100% containment at 0.05 microns upwards with the possibility that these size limits are updated to be more stringent with a size limit review. See:

• The European Space Foundation in 2012 said we have to contain the far smaller ultramicrobacteria and gene transfer agents
  - 100% containment at 0.05 microns or larger and 1 in a million for release of a single particle ever at 0.01 microns or larger
  - well beyond capabilities of a BSL-4
   
• The ESF says we need to do a new review of the size limit and level of assurance regularly - definitely needed a decade later - so even their limit is now out of date

Then, there are many issues with a ground-based telerobotic facility too, such as

• accidents,
• criminal damage,
• we have no experience of operating such a facility.
• How do we replace or repair equipment, and would it really prevent a lab leak?

Also, it may be a requirement that

• the complete facility can be sterilized when it’s decommissioned, for instance if we find we have returned mirror life.

Then there's the time to build it, to train people to use it and so on.

As an exercise, based on these considerations, I sketched out an idea for a telerobotic facility that would deal with all these issues at a very high level of assurance:

The aim here is simply to demonstrate that it may be technologically feasible not to minimize cost. There are many disused nuclear bunkers so that's a simple way to keep it very safe from impacts by aircraft, even in a warszone indeed. Then built inside an oven for sterilization when it's decommissioned. Then I looked at a way to have access to it with no risk of forwards or backwards contamination either way. This is what I came up with, a sketch of an idea.

JUST TO TRY TO ESTABLISH FEASIBLITY FOR 100% PROTECTION OR VIRTUALLY 100% FOR EARTH'S BIOSPHERE - NO ATTEMPT TO MINIMIZE COSTS.

Text on graphic: Sketch for 100% containment of mirror namobes etc. Sump kept at 300°C filled with Pentaine X2000 oil. Both airlocks and sump continuously radiated with X-rays and ionizing radiation and sterilized with CO2 snow. Both airlocks +ve pressure, inlets sealed during airlock cycles.

Shows the LAS fully robotic floor plan for a Mars sample receiving facility placed inside an oven for end of laboratory lifetime sterilization of the facility and accessed via two airlocks and a sump for 100% containment of even mirror life nanobes.
Sketch of telerobotic facility Credit NASA / LAS ( Keeping Mars Contained,) Photo of Cultybraggan nuclear bunker (Cultybraggan nuclear bunker) .

This is just a sketch of basic scientific ideas not an engineering proposal. It look as if this could be developed into a feasible solution. The sample could be returned to Earth in a titanium sphere since those survive re-entry undamaged ( Oregon Space Ball, probably from the equipment module of Gemini 3, 4, or 5 mission, titanium) , and it could be covered in a Whipple shield to protect against micrometeorite impact which might be foam based (Shield Development) and double as the shielding used for a black box flight recorder built to withstand even a plane crash (Black box flight recorders fact sheet), only opened inside the telerobotic facility.

I cover this in the attachment to my last comment, updated version here: NASA and ESA are likely to be legally required to sterilize Mars samples to protect the environment until proven safe...

It's hard to say how feasible or otherwise this is, as I found no research into a telerobotic facility to the standard required by the ESF sample return study - but it seems likely to be far more expensive than a small satellite launched in the early 2030s to above GEO, and needs more upfront funding in the 2020s.

If we find that Martian life is mirror life or in some other way so hazardous for Earth’s biosphere that it has to be contained far above a BSL-4, we may later need something larger and better resourced than a miniature telerobotic lab above GEO. But by then we likely have a permanent presence on the Moon. This is a decision for the future but the best solution may be a telerobotically controlled laboratory on the Moon.

Hazardous Biology Facility on the Moon, telerobotically attended, surrounded by vacuum - Artist's impression, illustration by Madhu Thangavelu and Paul DiMare © (The Moon: Resources, Future Development and Settlement : : 145, 146).

This would be a step up from the orbital satellite while keeping it 100% contained more easily than on Earth.

Humans would still go nowhere near the facility but in a future where we have a permanent human presence on the Moon we could operate such a facility far more easily than one in GEO, for instance to install new robotic handlers, to add / remove materials, to service and maintain it etc especially if by then we need a large facility with large multi-tone instruments. We could even build a particle accelerator or accelerators on the lunar surface for such a facility to use.

See below: We can eliminate all these issues and make it a far simpler mission using a miniature telerobotic facility above GEO

We can't use safety testing either in orbit or on Earth to prove the samples are safe to release unsterilized
- all Perseverance's samples would go to hold and critical review following the COSPAR Sample Safety Assurance Framework
- because of the terrestrial contamination at 8.1 ppb
- and because we don't have adequate knowledge of the terrestrial contamination sent to Mars
- well over 1000 genera with two few reads to identify them and only 54 genera identified
[IN NEPA BOTH]

Video: Open letter to NASA: Safety testing doesn't work even for clean samples above GEO

I found your “safety testing” wouldn’t work, either in orbit or on Earth. All unsterilized samples would go to “hold and critical review” for two reasons

• the high level of terrestrial contamination in the Perseverance samples,

In the COSPAR Sample Safety Assessment Framework, say that if life is detected it's not feasible to predict harmful or harmless consequences if life is detected: So safety testing consists only of testing to see if there is any life there.

.Unfortunately, we have only a limited ability to predict the effects of terrestrial invasive species, emerging pathogens, and uncultivated microbes on Earths' ecosystems and environments. This is true even for cultured and fully genome-sequenced terrestrial organisms and more so for potential extraterrestrial life. Thus, conducting a comprehensive sample safety assessment with the required rigor to predict harmful or harmless consequences of potential martian life for Earth is currently not feasible.

 

.Conducting a comprehensive safety assessment with the required rigor to predict harmful or harmless consequences for Earth is not feasible. Therefore, the scope of the SSAF is limited to evaluating whether the presence of martian life can be excluded in the samples. Any possible hazard is only considered in the sense that if there is no martian life, there is no extraterrestrial biological hazard in the samples.

(Kminek et al, 2022, COSPAR Sample Safety Assessment Framework (SSAF) )

But the high levels of contamination permitted by Perseverance would make that testing impossible.

One complication is that terrestrial biological contamination would impact the specificity of the test, that is, leading to a false positive.

...

(for step 7)

It is expected that this step could lead to a number of positive events that are likely associated with terrestrial contamination. However, until any evidence for life can be clearly associated with terrestrial contamination, the conservative assumption (positive hypothesis) is that it could be martian biology

(Kminek et al, 2022, COSPAR Sample Safety Assessment Framework (SSAF) )

To deal with that issue they refer to the need for contamination knowledge:

The contamination baseline for returned martian samples must be established from the CK [Contamination knowledge] obtained during the assembly of the various spacecraft that will fly as part of the MSR Campaign, along with blanks and witness samples returned with the martian samples

However as we saw above, we don't have that knowledge. Over 1000 genera were found that couldn't be read. Also the samples were very varied with for instance, 36 out of 49 spore forming species found in only one of the 98 swabs. See above:

• No way to reliably distinguish terrestrial and martian biosignatures - the attempt at a genetic inventory turned up over 1000 distinct genera but with too few reads to identify them - and amongst the 54 genera that could be identified, found four species with less than 98.7% resemblance to the closest known species
   
• The "witness tubes" which collect terrestrial contamination in a dummy run of sample collection can help geologists subtract contamination to get a good idea of the original levels of organics - but are not enough to reveal biosignatures when terrestrial contamination is likely orders of magnitude higher than biosignatures astrobiologists are interested in

The combination of the high level of contamination - and the varied composition of samples in the Perseverance rooms, would surely make it impossible to rule out terrestrial contamination.

Even 100% contamination knowledge wouldn't be enough for a high level of assurance
- suppose we send chroococcidiopsis to Mars
- then we find chroococcidiopsis in the returned sample
- this doesn't show it is safe unless we are certain its the same strain we sent to Mars from Earth
- a novel Martian strain of chroococcidiopsis evolved over billions of years on Mars could have new capabilities or novel toxins

Also - even if we found very familiar life, such as another strain of chroococcidiopsis on Mars, it could have developed novel capabilities in the very different conditions on Mars. It could have added

• a new metabolic pathway,
• more efficient photosynthesis,
• some new toxin,
• an undetected new fungal parasite
• undetected inherited prions
• could co-exist in the sample with undetected novel biology such as life based on mirror organics

Any of these could make it unsafe to return to Earth. It's especially likely that related strains on Mars could produce novel toxins. Microalgae produce many toxins. As an example, Chroococcidiopsis Indica produces BMAA, a neurotoxin which can cause Lou Gerig syndrome, the disease Steven Hawking had.(Diverse taxa of cyanobacteria produce β-N-methylamino-L-alanine, a neurotoxic amino acid) .(Warmflash et al, 2007, Assessing the Biohazard Potential of Putative Martian Organisms for Exploration Class Human Space Missions).

There are numerous other toxins expressed by cyanobacteria and often they depend on a gene carried by a particular strain. According to some estimates, 25% to 75% of cyanobacteria blooms are toxic in one way or another (Toxins produced in cyanobacterial water blooms – toxicity and risks)

"Each toxin is produced by cyanobacteria only when the appropriate toxin gene is carried by a particular strain and if its expression is activated by environmental conditions

Cyanotoxins are usually classified in four classes according to their toxicological target:

1. hepatotoxins that act on liver (Microcystins and Nodularin)
2. cytotoxins that produce both hepatotoxic and neurotoxic effects (Cylindrospermopsin)
3. neurotoxins that cause injury on the nervous system (Anatoxins, Saxitoxins and ß-Methylamino-L-Alanine –BMAA-) and
4. dermatoxins that cause irritant responses on contact (Lypopolysaccharide, Lyngbyatoxins and Aplysiatoxin

(Cyanotoxins: methods and approaches for their analysis and detection)

These novel toxins could also be transferred to other microbes via gene transfer agents. More generally, we can’t know that unsterilized samples from Mars are safe for Earth until we have a much better understanding of Mars’s biosphere, if any, know what to look for, what its capabilities are and so on.

Safety testing in an orbital facility can't be used at all if microbes occur in very low concentrations of only a few microbes per sample - even if, improbably, most of the sample was destructively tested for biosignatures, we couldn’t rule out a viable spore perhaps imbedded in a crack in a dust grain in the remaining fraction of the sample

The COSPAR Sample Safety Assessment Framework (SSAF) refers to this problem that there is no guarantee that any Martian life has got into the subsamples examined.:

There is also another complication: even if there is life somewhere in the sample tube, there is no guarantee that there will be life in the subsamples that are examined.

(Kminek et al, 2022, COSPAR Sample Safety Assessment Framework (SSAF) )

This issue could be very acute in some scenarios

• Scenario of life in the Martian dust at low concentrations, a few viable cells per gram or less, brought to Jezero crater from distant locations on Mars.

This was identified as a knowledge gap in the Space Studies Board review of SR-SAG2. The EIS uses SR-SAG2 but doesn't mention the Space Studies Board review of it, which was commissioned by NASA and ESA out of concerns that SR-SAG2

"The SR-SAG2 report does not adequately discuss the transport of material in the martian atmosphere. The issue is especially worthy of consideration because if survival is possible during atmospheric transport, the designation of Special Regions becomes more difficult, or even irrelevant."

Microbes could be transferred from distant parts of Mars and still be viable, for instance in fragments of biofilms (A desert cyanobacterium under simulated Mars-like conditions in low Earth orbit: implications for the habitability of Mars), They might also travel in bouncing sand grains (Wind-driven saltation: an overlooked challenge for life on Mars).

Sagan suggested a viable microorganism could be imbedded in a dust grain and be protected from the UV by the iron oxides in the dust (Contamination of Mars. : 8)

Billi et al made a similar suggestion:

… Our findings support the hypothesis that opportunistic colonization of protected niches on Mars, such as in fissures, cracks, and microcaves in rocks or soil, could have enabled life to remain viable while being transported to a new habitat
( A desert cyanobacterium under simulated Mars-like conditions in low Earth orbit: implications for the habitability of Mars)

The largest dust grains detected by Curiosity reached above 6 microns in diameter for 3 days during the 2018 dust storm (Large dust aerosol sizes seen during the 2018 Martian global dust event by the Curiosity rover : Figure 4 and discussion of it).

I can't find any experiments that test this hypothesis of microbial survival within dust grains with such large particles. If anyone reading this knows of such an experiment please let me know.

• Scenario of a random sampling of a few stray microbes from a nearby colony that Perseverance didn't detect.

Perseverance has no in situ life detection capabilities and Martian life could be mixed with the dust, dark in colour, and not obvious to the rover. Perseverance could randomly sample the very edge of a colony or just outside it where a few stray microbes might still be found, at similar concentrations - of just a few cells per sample tube or even only one viable cell per gram or less.

This issue of biological oases is especially acute in the most inhospitable places:

When searching for evidence of life, the probability of a false negative result is highest in environments where potential biosignatures occur at very low abundance (e.g., due to low productivity or to degradation/destruction processes), operating at a very low (or even dormant) metabolic state, or where life is not distributed homogeneously (i.e., biological oases amidst an abiotic landscape).

...

Biological oases typically occur in areas where resources (water, nutrients, energy) are locally more abundant, or where lethal environmental conditions (e.g., radiation, excessive temperatures) are somehow mitigated. Life signatures can be relatively diverse and abundant in those oases, but quickly vanish with distance or time. Often, biological oases are associated with specific substrates or physical environments (rocks, sediments, subsurface layers, fracture surfaces) whose chemical or physical properties provide a survival advantage to organisms. .

National Academies of Sciences, Engineering, and Medicine, 2022, "Origins, worlds, and life: a decadal strategy for planetary science and astrobiology 2023-2032." : 396)

The 2022 National Academy of Sciences cite goes on to say that we can learn more about where oases are likely to occur so we look for life in likely places to find it. However since Perseverance isn't an in situ life detection mission, if there are biological oases in Jezero crater it will only sample them fortuitiously.

So if we need a high level of assurance, we can't do safety testing even for bonus samples with no terrestrial contamination, because

• even if, improbably, most of the sample was destructively tested for biosignatures, we couldn’t rule out a viable spore perhaps imbedded in a crack in a dust grain in the remaining fraction of the sample.

We can’t know that unsterilized samples from Mars are safe for Earth until we have a much better understanding of Mars’s biosphere, if any, know what to look for, what its capabilities are and so on.

We should be able to sterilize even unknown Martian exobiology by using levels of ionizing radiation that will break the covalent bonds that hold complex chemicals together
- 100 million years equivalent of surface ionizing radiation may be sufficient but this needs expert attention in a multi-disciplinary approach involving experts in synthetic life and possibilities for alternative evolutionary pathways
- no effect on isotope ratios and virtually no changes for geology

We should be able to sterilize even unknown Martian exobiology by using levels of ionizing radiation that will break the covalent bonds that hold complex chemicals together - 100 million years equivalent of surface ionizing radiation may be sufficient but this needs expert attention

This needs research into the level of ionizing radiation needed. We could use high levels with almost no impact on geology as most of the samples will have had hundreds of millions or billions of years of surface ionizing radiation, well beyond any sterilizing dose for terrestrial life.

Ionizing radiation has little effect on geology - salt crystals change colour but at doses far less than expected for even recently exposed materials on Mars, no changes in crystal spacing and no changes in the ratios of isotopes for radiometric dating after 3 million years equivalent of ionizing radiation (Allen et al, 1999, Biological sterilization of returned Mars samples).. We likely need much more than 3 million years, but no reason to expect any change in isotope ratios and most or all of the specimens except the surface soil / dust / salts would likely have had far more ionizing radiation dose already.

We should be able to sterilize biological entities even if not based on terrestrial biology

It is believed that if such a biological entity exists, humans would be able to kill it (by the sundering of covalent bonds in a rigorous sterilisation process).

(Ammann et al., 2012, Mars Sample Return backward contamination–Strategic advice and requirements : 12)

The main question is, what is the sterilizing dose needed? There doesn’t seem to be much research on the levels of ionizing radiation needed to sterilize any present day life on Mars if it is of some completely different biology. Rummel et al. say that the bond breakage should be similar if it’s based on other than carbon-containing molecules

If there were a life-form on Mars based on other than carbon-containing molecules, the energies holding such molecules together would not be much different than those for proteins and polynucleotides.

Hence, bond breakage by heat or gamma radiation should be similar for Earth and Mars life-forms, and sterilization conditions for Earth microorganisms should eradicate microorganisms of similar size from Mars.

There is no absolutely optimal approach to decontamination under these circumstances, but enough is known about the relationships among organism size, repair mechanisms, and survivability, that the maximum survivability of any martian organisms can be estimated with some confidence.

It is believed that if such a biological entity exists, humans would be able to kill it (by the sundering of covalent bonds in a rigorous sterilisation process).

Irrespective of the chemical basis of any life-form, a confidence level of sterilization can be provided with only two assumptions: 1) any reproducing life-form must be based on macromolecules (i.e., polymers) with interatomic covalent bonds (not crystal lattices), and 2) since all such bonds have similar strength, destroying these bonds destroys the life-form.

(Rummel et al., 2002, A draft test protocol for detecting possible biohazards in Martian samples returned to Earth : 10, 13)

Let's just look briefly at that first assumption (not crystal lattices). There's an intermediate possibility, the question of whether we need to sterilize prion aggregates - which only repeat in one direction. They aren't alive but they are able to replicate in living organisms which provide them with a supply of proteins to add to them. There is a change since the 1997 report, we now know of prions in microbes in all three of the domains of life, archaea, bacteria and eukaryotes, so they likely also occurred in the last universal common ancestor:

• A new size limit review could look at prions - raised by the biosafety report and last considered in 1997 because of the very recent discovery of prions in all three of the domains of life, archaea, bacteria and eukaryotes and so probably in the last common universal ancestor too [NEW]

Prions are proteins in their minimum energy state which lets them form amyloid fibres based on β-sheets. These consist of lots of parallel proteins in a stretched out low energy configuration, bonded together by hydrogen bonds.

(Amyloid of Rnq1p, the basis of the [PIN+] prion, has a parallel in-register β-sheet structure. 2008 : Figure 6) (PNAS license)

If we need to sterilize these structures we need higher sterilization doses than for DNA which needs a double strand break to break it up, or for protein polymers which can be broken into pieces by cutting them in one place.

Then for crystal structures themselves, there's Cairn-Smith's suggestion in his book from 1985 (Seven clues to the origin of life: a scientific detective story) that life might have started based on a crystal lattice of clays and replicating clay sheets that form on the surface and then flake off, before it developed cell walls and cell processes. Clays could propagate as defects in one layer can lead to defects in the next (Astrobiology: A Brief Introduction: 127). Cairn-Smith's idea originally seemed promising as an idea, with a lot about it that is in common with the way that DNA works. However experiments don't seem to support it, though they led to other interesting ideas for ways that clay could have helped seed some of the processes of early life (Clays and the origin of life: The experiments., 2022) .

Cairn Smith's theory was to explain present day life, so it starts 2D and then shifts to a 1D polymer and the modern DNA helix. For a life-form that keeps its genetic information in a 2D lattice it would need to remain 2D or else start from 1D and shift to 2D. If alien life could find a way to achieve high fidelity replication from one sheet to the next in 2D, and to store its genetic information in sheets rather than in helixes, it would be a simpler form of replication, and with higher density of information so it might have advantages.

There doesn't seem anything to support this as a possibility. I'm just mentioning it for completeness here. Do we need to consider it? If we need to consider 2D lattices It would still be possible to sterilize alien life by breaking the bonds - but it would take a higher dose to break up 2D sheets into discontinuous molecules, especially so if they were stored in stacked identical sheets.

I cover this in my preprint, concluding:

I find little in the literature by way of attempts to estimate how more resilient a totally alien biochemistry might be, evolved to resist ionizing radiation on Mars for billions of years. We can surely find a dose that would sterilize even a mirror life microbe with a PNA backbone adapted to Mars for billions of years. Is 10 megagrays enough, or 100 million years equivalent?

This is something that needs attention from experts in the relevant disciplines such as synthetic biology, early life, etc.

NASA must protect Earth's biosphere even if Mars samples hold mirror life ...) : Section: "Sterilization of the samples must be effective for any conceivable exobiology – 100 million years equivalent would reduce DNA strands through double strand breaks to fragments only 29 bases long though alien biomolecules such as PNA might be more resilient")

Anyway this topic needs to be looked at carefully, but it seems reasonable that we can come up with a level of sterilization that everyone is confident will work. The aim is to find a level of sterilization we can have confidence in generally.

For the simplest alternative we just sterilize all samples returned from Mars with virtually no effect on the geology and probably virtually no impact on the astrobiology if we don't add th bonus samples.8 of the public already suggested the alternative of a sterilized sample return in the first round of public comments and it should be included as an alternative alongside the obligatory no action for the full range of options.

• sterilize first – sterilize or test first – sterilize first – sterilize first – sterilize first - sterilize first, test method of sterilization on Mars life in situ

Plus my comment:

• (Walker, 2022, Comment by posted on May 16th)

and

• (Dehel, 2022, Comment posted May 4th)· (suggestion in his attached file)

However we can achieve far better science with the miniature life detection lab idea.

We can eliminate all these issues and make it a far simpler mission using a miniature telerobotic life detection lab above GEO - for ALL the work with unsterilized samples including life detection and astrobiological research - NOT for safety testing - by 2033 we should be able to send all the instruments iMost recommended for present day life to orbit, in miniature [IN NEPA BOTH]

But we now have the capability to set up a miniature telerobotic facility just a few meters across above GEO due to the amazing shrinking in size of astrobiological life detection instruments designed for future in situ missions to Mars. Every decade the new instruments are even smaller.

Some are ready to fly today. Others are at an earlier stage of technological readiness but being actively developed, and may well be ready by the 2030s, and especially so if there is a strong incentive to complete them. Here are a couple of interesting examples:

• Life Marker Chip LDChip300 (antibodies) almost sent on Exomars but descoped which was able to discover a previously undetected microhabitat in the Atacama desert
  (A microbial oasis in the hypersaline Atacama subsurface discovered by a life detector chip: implications for the search for life on Mars)
  target mass of less than 1 kg (Life Marker Chip)
   
• Gene sequencer SETG (Search for Extraterrestrial Genomes project) designed to do end to end sequencing on Mars using the MinION nanopore sequencing technology. By 2016 it reached the capability to detect 10 ppb of DNA. It would be able to detect 400 highly conserved “universal genes” such as ribosomal DNA which would establish whether Martian life is related to terrestrial life, perhaps by meteorite transfer, or is independently evolved (Mojarro et al., 2016, SETG: nucleic acid extraction and sequencing for in situ life detection on Mars).
  
  SETG needs more work but is being actively explored by NASA for in situ detection for icy worlds (Carr et al., 2020, Nanopore sequencing at Mars, Europa, and microgravity conditions). It uses biodegradable organic nanopores at present with a short shelf-life, and NASA are exploring non biodegradable nanopores for longer missions to Mars and icy moons of Jupiter and Saturn, which would also help with issues of resupply of the nanopores to above GEO. They are investigating “nanogaps”, solid state nanopores that can also detect and distinguish amino acids - so they may be able to sequence proteins too (Maggori, 2023, Life detection and taxonomic characterization with MinION sequencing in Mars and icy worlds analogue environments : 230)

NASA's iMOST report was published too soon to take advantage of the Europa lander report and it missed many instruments that already existed for in situ study

See: We DO have the instruments available to do ultrasensitive measurements to search for life above GEO
- your own study for Europa from 2016 and published in 2017 says so
- but your iMost study was too early to take account of its results
- and also missed many in situ instruments already designed

I have a list of many more of these amazing shrinking instruments towards the end of this open letter. See:

• NASA already have worked on plans for a remote life detection laboratory on Europa in the report of the Europa Lander Science Definition Team
  - numerous shrinking exquisitely sensitive life detection instruments
  - whatever we can do using a remotely operated lander on Europa
  - we can do in a far easier fashion in a small telerobotic facility above GEO
  - with a latency of a quarter of a second return trip [IN NEPA BOTH]

They would be able to make progress with all the measurements envisioned by the iMost team for present day life.

Text on graphic: Bonus samples in STERILE containers returned to satellite perhaps 50,000 or 100,000 km above GEO in what would be Earth’s ring plane if it had a ring system.

• NOT for safety testing
• Returned for astrobiological study – nexus of expanding off-planet astrobiology lab.
• Minimal forward contamination.
• Humans nowhere near this.
• Centrifuge to replicate martian gravity.

Many instruments placed in centrifuge along with the dust and operated remotely from Earth. Illustration shows:

• Chiral labeled release.
• SETG from sample acquisition through to DNA sequence all automated in 2 units, each can be held in palm of hand.
• Astrobionibbler microfluidics can detect a single amino acid in a gram of sample

This would be minimal cost for NASA as the instruments would be funded by universities.

Graphic shows: (NOAA’s new GOES-17 weather satellite has degraded vision at night) just to have an image of a geostationary satellite, not that it would be a $2.5 billion dollar satellite. SETG from (Mojarro et al., 2016, SETG: nucleic acid extraction and sequencing for in situ life detection on Mars). Astrobionibbler from (Elleman, 2014, Path to Discovery) ISS centrifugal motor for plant experiments, dialable to any level from microgravity to 2g (Centrifuge Rotor [biology experiment on the ISS])

The ISS has a centrifuge and it uses "Slip Ring Technology" to supply air, water and power to the rotating portion. So the experiments inside the centrifuge can be powered from outside. the axis.

The CR development involves the challenge of "Slip Ring Technology" that bridges the rotating portion and the static portion of the CR. It supplies air, liquid and power to the rotating portion of the CR and receives video signals from the rotating portion to the static portion.

(NASA, n.d.,(Centrifuge)

This life detection lab above GEO would double as an excellent Mars simulation chamber better than the BIOMEX experiment which ran on the exterior of the ISS

This is BIOMEX - it simulated the UV on Mars by slightly filtering the light from the sun. It simulated the ionizing radiation because the ionization radiation levels are similar to those on Mars for the ISS (would be somewhat higher above GEO but should still be okay for microbes adapted to Martian conditions). Was easy to simulate the Martian atmosphere.

(Exposed! International Space Station Tests Organisms, Materials in Space) (Limits of Life and the Habitability of Mars: The ESA Space Experiment BIOMEX on the ISS)

This time it would have artificial Mars gravity - and for a more realistic simulation, the satellite could have the centrifuge axis constantly pointing towards the sun, with day and night and seasons simulated by moving sliders to block out the sunlight during the simulated nights, and perhaps humidity also varied seasonally to simulate Jezero crater conditions. This could be done relatively easily in space.

The centrifuge could use glass windows similarly to BIOMEX on the axis. Or perhaps it could use solarization resistant optical fibers to transmit sunlight from outside the satellite along the axis and into anywhere within the centrifuge. These are designed to transmit UV with much less formation of colour centers that reduce transmission of UV, usually designed for much higher UV levels and almost no degradation over time periods of hours, would need fiber with similar properties operating for many hours a day but with night to recover . Either way we can get excellent replication of the solar flux on Mars as for BIOMEX.

Solarization refers to the formation of color centers within a fiber that lead to transmission degradation. These color centers form when exposed to light below 300 nm. Solarization-resistant fibers are thus desirable when working in the UV due to their superior transmission and prolonged performance. Typical applications for these fibers are spectroscopy and medical diagnostics. High-OH fiber experiences significant transmission loss when exposed to UV radiation. In contrast, solarization-resistant fiber offers higher transmission at these UV wavelengths. For optimal performance, expose the fiber to UV radiation for 5 minutes prior to use in your application to allow initial degradation. After this time, equilibrium is reached and the fiber can be used normally.

(Thorlabs, n.d., Solarization-Resistant Multimode Fiber Optic Patch Cables)

We can use a very safe orbit in the inclined Laplace plane far above GEO
- where Earth's ring particles would orbit if it had a ring system
– about as far as you can get in delta v from Earth or the Moon in cislunar space
- and in the same plane as a proposed disposal orbit for geostationary satellites
- even high area to mass ratio debris (HAMR) from the new satellite, such as insulation debris, can't contaminate satellites in GEO

We can target the Laplace plane inclined at approximately 7.2° from the equatorial plane. This is in the same plane as a proposed “graveyard orbit” for GEO satellites at end of lifetime as even large light fragments of cladding from the satellites stay trapped well away from GEO, through the balance of the light pressure from the sun and gravity . where ring particles would orbit if Earth had a ring system.

. The classical Laplace plane as a stable disposal orbit for geostationary satellites.

The sterilizing sample could be placed, say, 50,000 km or 100,000 km above this proposed GEO disposal orbit. This is very safe as the delta V is over 1 km / second to both Earth and the Moon and it would also be safe for GEO and far from the proposed Laplace plane GEO graveyard orbit.

2014. Access to Mars from Earth–Moon libration point orbits: manifold and direct options. A

One way is to return the sample via a lunar retrograde orbit (actually a prograde orbit around Earth but retrograde around the Moon). Lock et al. proposed returning a sample from Mars using a close flyby of Earth followed by a flyby of the Moon which then can get captured in a lunar retrograde orbit with a delta v of only 100 meters per second . First they need to reduce the speed of the spacecraft relative to Earth on the flight back, to make this capture easier. This increases the fuel mass by about 30% for their hypothetical mission. Assuming a 1000 kg dry mass t under 370 kg for the direct flight with aerocapture, this increases to a fuel mass of just under 480 kg for the low energy transfer ( Mars Sample Return Orbiter Concepts Using Solar Electric Propulsion for the Post-Mars 2020 Decade)

From the lunar retrograde orbit, it is easy to transfer to Lunar L2 (LL2) ( . Exploration of distant retrograde orbits around Moon), the gravitational point of balance beyond the far side of the Moon as seen from Earth. From there it can do a low energy transfer to a Lunar L1 halo between Earth and the near side, and then it can get to above GEO, using lunar flybys in fuel efficient ballistic transfer trajectories again, to reduce the total delta v requirements.

Another possibility goes via the Sun Earth L2 then dovetails to the Lunar L2 ( Access to Mars from Earth–Moon libration point orbits: manifold and direct options. : figures 13 and 10 in reverse) - this avoids even close flybys of Earth or the Moon although flybys are now well established technology and don't pose any risk of impact.

The delta v to send payloads to 100,000 km above GEO is almost identical to the delta v for GEO. I did a little online calculator to help, as a first rough idea.

Missions start in LEO at altitude 250 km and velocity 7.755 km / sec.

Transfer to GEO at altitude 35786.154 km and velocity 3.075 km / sec:
Delta V = 3.912 km / sec = 2.44 km / sec (leave LEO) + 1.472 km / sec (insertion to GEO).

Transfer from LEO to orbit at altitude 100000 km and velocity 1.936 km / sec:
Delta V = 4.158km / sec = 2.886 km / sec (leave LEO) + 1.273 km / sec (insertion to orbit at 100000 km).

Transfer from LEO to the orbit at altitude 100000 km needs:
Extra delta V = 0.246 km / sec
- compared to the transfer to GEO.

(Calculator to find the delta V to get from LEO to GEO, or from LEO to a higher orbit, and calculate the difference between the two delta v's.)

The “Earth Entry Vehicle” can be converted into an “Above GEO Insertion Vehicle” by replacing the aeroshell with extra fuel
– and returned to this orbit without concerns about aerobraking
– we can use the EEV’s ion thruster for low energy ballistic transfer

We need to avoid aerobraking and we can do that using “ballistic capture”, also known as “weak stability boundary transfers” (Topputo, et al., 2015. Earth–Mars transfers with ballistic capture), the low delta v, fuel efficient, three or four body transfer orbits first used for the Japanese Hiten mission in 1990 (Belbruno,, 2018. Capture dynamics and chaotic motions in celestial mechanics: With applications to the construction of low energy transfers). The ESA Earth Return Orbiter will use continuous low thrust transfer (Hurowitz et al., 2017. Redox stratification of an ancient lake in Gale crater), ideal for ballistic capture.

One way is to return the sample via a lunar retrograde orbit (actually a prograde orbit around Earth but retrograde around the Moon). Lock et al. proposed returning a sample from Mars using a close flyby of Earth followed by a flyby of the Moon which then can get captured in a lunar retrograde orbit with a delta v of only 100 meters per second . First they need to reduce the speed of the spacecraft relative to Earth on the flight back, to make this capture easier. For their hypothetical mission this increases the fuel mass by about 30%. Using the example of a 1000 kg dry mass and fuel mass of just under 370 kg for the direct flight with aerocapture, this increases to a fuel mass of just under 480 kg for the low energy transfer with the same dry mass ( Mars Sample Return Orbiter Concepts Using Solar Electric Propulsion for the Post-Mars 2020 Decade). That's about 1.1 kg of extra fuel compared to a dry mass of 1000 kg from which we can subtract the mass of the aeroshell and also subtract the mass of the fuel needed to transport the aeroshell to Mars and back.

From their lunar retrograde orbit, it is easy to transfer to Lunar L2 (LL2) ( . Exploration of distant retrograde orbits around Moon), the gravitational point of balance beyond the far side of the Moon as seen from Earth. From there it can do a low energy transfer to a Lunar L1 halo between Earth and the near side, and then it can get to above GEO, using lunar flybys in fuel efficient ballistic transfer trajectories again, to reduce the total delta v requirements.

This does need a little extra fuel compared to the direct flight and aerocapture for the same amount of dry mass. However, it also saves the mass of the aeroshell, which can be replaced by fuel. Also the saving on the aeroshell saves on fuel both on the journey to Mars and the journey back. So, it's not clear that more fuel is needed overall depending on the mass of the aeroshell, and if more fuel is needed, it is for a lower dry mass, so the total mass may still not be changed or even reduced. This needs analysis to see if it does change the total launch mass, and if so, if it is an increase or even a reduction in total mass.

The flybys of Earth and the Moon are now well established technology and don't pose any appreciable risk of impact so long as the spacecraft is trajectory biased away from Earth, and never is on a course that would let it hit Earth at any point.

Another possibility goes via the Sun Earth L2 then dovetails to the Lunar L2 ( Access to Mars from Earth–Moon libration point orbits: manifold and direct options. : figures 13 and 10 in reverse) - this avoids even close flybys of Earth or the Moon.

This keeps Earth 100% safe with virtually no loss to science and little change in NASA’s budget
– adds the cost of a Sample Sterilizing Satellite but saves on the mass of the aeroshell and the cost of a Sample Receiving Facility

With this option, NASA has the extra cost of the sterilizing satellite, but save on several other costs including the cost of a sample receiving facility on Earth. Even a normal BSL-4 involves significant costs. Assuming it's agreed that we can't protect human health or Earth's biosphere from microbes with unknown capabilities using quarantine of technicians, a fully telerobotic facility to contain the samples on Earth would likely cost far more than a miniature life detection lab above GEO.

For the cost saved for the sample receiving facility, it would be possible to send a large satellite to geostationary orbit. Also universities might well be interested to join in on the cost.

The sample receiving satellite could also be a special item for a NASA budget in Congress assuming public interest in protecting Earth. If NASA explains to Congress that they need to protect Earth and that this is a way to do it then that may unlock extra funding here.

But it's not clear we need any extra funding relative to the current mission concept.

As for powering the experiments and other operations, a satellite placed above GEO can have many kilowatts of power available from solar panels. The Inmarsat 5 F1 has a power supply of 15 kilowatts on launch (Inmarsat, 2013, Successful launch confirmed for Inmarsat’s first Global Xpress satellite (Inmarsat-5 F1))

We can expect much of the cost of the instruments sent to above GEO to be found by universities interested in testing their own designs of in situ instruments to search for life on Mars.

In this way we keep Earth 100% safe, with virtually no loss to science and little change in overall budget.

Rough estimate of payload capacity to above GEO
- with the Falcon Heavy allowing for station keeping for 15 years, and transfer from GTO, we should be able to send 20 tons at a cost of less than $100 million
- with the Atlas V 541 as for Perseverance we can send nearly 5 tons to above GEO
- compare 1.025 tons for Perseverance,
- and even more capacity by 2033
- so there isn't any issue with sending numerous small life detection instruments to above GEO

For the launch costs to above GEO, the Falcon 9 can deliver 8.3 tons to Geostationary Transfer Orbit (GTO) at a cost of $67 million and the Falcon Heavy can already deliver 26.7 tons to GTO at a cost of $97 million for the reusable rocket (SpaceX, n.d., Capabilities and Services).

We have to allow for transfer from GTO to above GEO and for station keeping. Although we likely wouldn't send anything this large we could send multi-ton satellites to above GEO. With a rough calculation, I make it about 5 tons dry mass allowing for fuel and station keeping if we use the Atlas V, to send to above GEO,  compared to 1.025 tons for Perseverance. It's over 20 tons if we use the Falcon Heavy, in both cases using electric propulsion. The launch costs even for the Falcon Heavy at over 20 tons to above GEO would be less than a fifth of a fully telerobotic terrestrial facility.

The Falcon Heavy's 26.7 tons gets reduced because a satellite needs to use some of its mass to circularize to GEO, and by a little more because the destination is above GEO. But we can expect to deliver over half of the GTO mass to our final orbit above GEO, or over 14 tons. If the spacecraft uses electropropulsion to transfer from GTO to GEO we may be able to deliver over 20 tons to GEO using the Falcon Heavy. Then we need to allow for station keeping.

There is very little by way of extra delta v to transfer to a higher orbit at say 100,000 km above GEO. I did a little online calculator to help, as a first rough idea.

Missions start in LEO at altitude 250 km and velocity 7.755 km / sec.

Transfer to GEO at altitude 35786.154 km and velocity 3.075 km / sec:
Delta V = 3.912 km / sec = 2.44 km / sec (leave LEO) + 1.472 km / sec (insertion to GEO).

Transfer from LEO to orbit at altitude 100000 km and velocity 1.936 km / sec:
Delta V = 4.158km / sec = 2.886 km / sec (leave LEO) + 1.273 km / sec (insertion to orbit at 100000 km).

Transfer from LEO to the orbit at altitude 100000 km needs:
Extra delta V = 0.246 km / sec
- compared to the transfer to GEO.

(Calculator to find the delta V to get from LEO to GEO, or from LEO to a higher orbit, and calculate the difference between the two delta v's.)

Based on a 15 year mission, with chemical motors and station keeping, the dry mass is 27% of the initial mass including fuel which would be 7.2 tons. If we can use electric propulsion then the dry mass is 61% of the initial mass or 16.28 tons. These are illustrative figures based on a worked example of transfer from GTO to GEO in:(Thomas, 2016, A Comparison of GEO Satellites Using Chemical and Electric Propulsion : Table 2). If we use the Falcon 9 and the 61% figure, the 8.3 tones is reduced to a little over 5 tons to GEO.

It's reduced a little to target above GE and we also need to allow for returning the sterilized samples to Earth which eats into that 5 tons / 22 tons payload to GEO with electric propulsion. However, by comparison Perseverance has a dry mass of 1.025 tons and was launched on an ATLAS 541 (NASA, 2020, Perseverance launch) which can send a payload of 8.29 tons to above GEO (ULA, n.d., ATLAS V), almost identical to the Falcon 9 and almost identical 5 tons to GEO

So, based on that example anyway, a satellite in our safe orbit well above GEO can be around five times heavier than anything we can send in situ to Mars.

So, though we likely don't need anything as large as this, we could send something as large as five tons to above GEO if it's needed, at launch costs of a fifth or less of the at least half a billion dollars for a fully telerobotic sample receiving laboratory.

As we approach the 2030s, launch costs are sure to go down further. SpaceX might be flying the super-heavy by the 2030s. We may be able to send multiple emissions to the satellite at very low costs compared to today.

NASA already have worked on plans for a remote life detection laboratory on Europa in the report of the Europa Lander Science Definition Team
- numerous shrinking exquisitely sensitive life detection instruments
- whatever we can do using a remotely operated lander on Europa
- we can do in a far easier fashion in a small telerobotic facility above GEO
- with a latency of a quarter of a second return trip [IN NEPA BOTH]

NASA has already explored the idea of sending a life detection suite to Europa and many of the instruments mentioned here come from that report. which says:

The Europa Lander mission concept is designed to achieve ground-breaking science. The SDT (Science Definition Team) is confident that a payload matching or exceeding the requirements described herein could potentially reveal signs of life on Europa (Hand et al., 2017, Report of the Europa Lander Science Definition Team : xi).

The Europa lander team also had a self-imposed restriction on the life detection instruments they permitted to be included as it set the requirement that they ALL must be dual purpose, able to provide valuable information about the chemistry or geology even if there is nothing found of biological interest.

"Life-detection experiments should provide valuable information regardless of the biology results"

This meant they couldn't include instruments that are only of interest for searching for life, such as tests for microbial respiration though they could include experiments that were dual purpose that could also be used for geological studies. A miniature lab above GEO wouldn't be restricted in this way, we can send instruments to the lab specifically designed to search for life which would tell us nothing of geological or chemical interest. It would also have the advantage that materials such as headspace gases can be returned to Earth sterilized for further analysis.

Text on graphic: NASA's proposed lander to do in situ life detection on Jupiter's moon Europa

The proposed reasonable alternative is to do life detection for the samples from Mars in a small centrifuge in a telerobotic lab above GEO with round trip latency of ¼ sec instead of over an hour - where we can send extra payloads to replace / repair / upgrade instruments or add new instruments to follow up on discoveries made in orbit

Graphic from (NASA, 2017, Europa Lander Study 2016 Report)

This orbiting astrobiology lab is the equivalent of one geostationary satellite far above GEO. Humans can study the dust, dirt and atmosphere as they would on Mars using exquisitely sensitive in situ instruments designed for end to end sample preparation to analysis – these already exist such as the

• Life Marker Chip LDChip300 (antibodies) almost sent on Exomars but descoped which was able to discover a previously undetected microhabitat in the Atacama desert
  (Parro et al, 2011 A microbial oasis in the hypersaline Atacama subsurface discovered by a life detector chip: implications for the search for life on Mars)
  target mass of less than 1 kg (Life Marker Chip)
• the gene sequencer SETG designed to do end to end sequencing on Mars with obviously no preparation on Earth. By 2016 it reached the capability to detect 10 ppb of DNA. It would be able to detect 400 highly conserved “universal genes” such as ribosomal DNA which would establish whether Martian life is related to terrestrial life, perhaps by meteorite transfer, or is independently evolved
  (Mojarro et al, 2016, SETG: nucleic acid extraction and sequencing for in situ life detection on Mars)
• astrobionibbler able to detect a single amino acid in a gram
  (Searching for Organics in a Nibble of Soil )
  (Noel et al., 2016, In Situ Microfluidic Subcritical Water Extraction of Amino Acids)
  (Elleman, 2014, Path to Discovery)
• a chiral version of the Viking labeled release experiment
  (Anbar et al., 2012, A Chiral Labeled Release Instrument for In Situ Detection of Extant Life)
• An off-axis holographic microscope to let the focus be adjusted after the image is taken making it easier to image individual microbes in a liquid medium
  (Lindensmith et al, 2016, A submersible, off-axis holographic microscope for detection of microbial motility and morphology in aqueous and icy environments)
  - which doesn't need a lens so can be included on a chip in microfluidics systems
  

  "Extremely small, robust, low-power microscopes have been constructed using light-emitting diodes in an in-line DIHM geometry. This type of design is lensless and may be coupled to microfluidics to create compact on-chip systems. The advantages of this approach are very low mass and low power, insensitivity to alignment, and lack of mechanically sensitive optics. Field of view is also decoupled from resolution because there is no objective lens.
  
  The field of view is 24 mm² without scanning and the entire instrument weighs < 100 g ..
  
  Super-resolution has been achieved in these systems using LEDs arrays where each is sequentially illuminated ... and a corresponding frame of data captured. In this case, the image from each LED is a slightly shifted hologram; the Fourier representations may be summed to achieve ~800 nm spatial resolution."
  Nadeau et al., 2018. Imaging technologies and strategies for detection of extant extraterrestrial microorganisms : 235)

• A method to check for redox reactions directly by measuring the electrons and protons they liberate. This is sensitive to small numbers of microbes and has the advantage that it could detect life even if not based on carbon or any form of conventional chemistry we know of
  (Ximena et al, 2010, Microbial fuel cells applied to the metabolically based detection of extraterrestrial life)
• A miniature variable pressure electron microscope that combines imaging with in situ chemical analysis
  (Gaskin et al, 2012, Miniature variable pressure scanning electron microscope for in-situ imaging & chemical analysis)
  
  This would be especially useful if we have already identified life with the other instruments and can bring candidates into the field of view of the microscope, or can cultivate martian life
  
  

  "Sample throughput is a major drawback of electron microscopy – the imaged volume is so small that it would not be useful for the ‘search’ part of a life detection instrument, though if candidate objects could be identified and brought into the field of the EM it could be valuable for evaluation of features indicative of life."
  Nadeau et al., 2018. Imaging technologies and strategies for detection of extant extraterrestrial microorganisms : 237-8)

    
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Several instruments suggested for Europa:

• Tests for autofluoescence. Aromatic amino acids (incorporating a ring of six carbons) fluoresce when stimulated with deep UV at wavelengths less than 250 nm. Chlorophyll and some other biological organics also autofluoresce
• We could also use fluorescent dyes that bond to specific macromolecules such as lipids, proteins and nucleic acids
• We can also use this autofluorescence to directly search for the activity of swimming microbes
• Raman microspectroscopy synchronized with visible light can do a chemical analysis of the microbes directly
• Superresolution optical microscopy, which can go beyond the usual optical resolution limit of 200 nm to observe nanobacteria

(Hand et al., 2017, Report of the Europa Lander Science Definition Team)

NASA's iMOST report was published too soon to take advantage of the Europa lander report and it missed many instruments that already existed for in situ study

See: We DO have the instruments available to do ultrasensitive measurements to search for life above GEO
- your own study for Europa from 2016 and published in 2017 says so
- but your iMost study was too early to take account of its results
- and also missed many in situ instruments already designed

So we are not in reality restricted to terrestrial labs. We can do all these experiments in situ on Mars with the advantage that the life is in its natural habitat or only recently removed from it - some microbes might die on the 6 months journey to Earth in darkness in a sample tube at low temperatures.

Astrobiologists are keen to send these missions to Mars but they seldom get selected and are always later descoped when they do. The main issue is that they are not tested in space. So we can test them in space in this small satellite above GEO first, searching for life in the samples. Then we can use them in Mars in the future. Meanwhile these are tiny instruments of a mass of order single digit kilograms typically with modern technology - and we could send hundreds of them in a ton of equipment. We could send far larger instruments to a satellite above GEO.

For experiments we can't do in orbit - such as analysing evolved gases for respiration experiments for hydrogen and nitrogen isotopes - perhaps we can do the experiments in orbit and return sterilized materials for analysis to Earth?

Most of the studies astrobiologists want to do with their samples can be done using instruments in situ on Mars and so could also be done in the orbiting satellite. This is another list of the studies they want to do from the 2019 conference for the search for extant life (Mars Extant Life: What's Next? Conference Report. ( html) :8022)

Figure 51 : from (Mars Extant Life: What's Next? Conference Report. ( html) :fig 10) adapted by Mackelprang from ( . Schrödinger’s microbes: tools for distinguishing the living from the dead in microbial ecosystems : fig 1)

Only the ones in bold boxes in this graphic currently need to be done on Earth . .

It should be possible to do the genome sequencing in the near future with SETG as we saw. We can do some analysis of the isotope ratios of headspace gases as for the analyses of the Martian atmosphere by Curiosity (Mahaffy et al.., 2013. Abundance and Isotopic Composition of Gases in the Martian Atmosphere from the Curiosity Rover). We can also store the gases, sterilize them, and send them back to Earth for testing, and perhaps some of the staining and microscopy could be done with sterilized samples from the growth medium sent back to Earth.

See: iMost propose to redo the Viking labeled release experiment
- but with far more sensitive measurements available today and labeling hydrogen (with deuterium) and nitrogen (with nitrogen 17) as well as the carbon
- some of this could be done in orbit
- some would need analysis back on Earth but perhaps the experiment itself can be done in orbit

For the microscopy, we do also have a holographic microscopic imager which will make it possible to change focus to observe swimming microbes with the returned 3D images, a scanning electron microscope, superresolution microscopy and synchronizing Raman spectroscopy with optical microscopy as we saw in the previous section.

So it does seem we can do almost everything - but for ultrasensitive studies of the headspace gases, especially for the non radioactive nitrogen and hydrogen isotopes we would need to return samples to Earth and also for samples that need extensive preparation that we can't yet do remotely.

So - there are some things we can't do in orbit. But we can do workarounds as much as possible and the upside is we have 100% protection of Earth. Also, we have many experiments that have a good chance of finding life if it is there.

Sterilization has no effect on isotope ratios.

• We should be able to sterilize even unknown Martian exobiology by using levels of ionizing radiation that will break the covalent bonds that hold complex chemicals together
  - 100 million years equivalent of surface ionizing radiation may be sufficient but this needs expert attention in a multi-disciplinary approach involving experts in synthetic life and possibilities for alternative evolutionary pathways
  - no effect on isotope ratios and virtually no changes for geology

iMost propose to redo the Viking labeled release experiment
- but with far more sensitive measurements available today and labeling hydrogen (with deuterium) and nitrogen (with nitrogen 17) as well as the carbon
- some of this could be done in orbit
- some would need analysis back on Earth but perhaps the experiment itself can be done in orbit

The Viking labeled release experiment worked by feeding the samples with organics labeled with radioactive carbon 14, Then it tested for radioactive carbon in the evolved gases.

The LR procedures for the Viking landers ,, measured evolved 14 CO 2 gas given off by 14 C-labeled carbohydrates reacting with constituents of the martian regolith. The widespread importance of the Krebs cycle in aerobic metabolism and the Embden–Meyerhof pathway in anaerobic metabolism makes the use of 14 C-labeled metabolites highly efficient in detecting microbial metabolism because both pathways produce carbon dioxide

The results gave “classic” biological responses for the two active cycles, and a negative response for the heat-sterilized cycle (cycle #2). These results were accepted as strong evidence for biological activity in martian regolith by Levin and Straat.

The controversy on this interpretation is based on two key points: (i) following a 2nd injection of the 14 C-labeled nutrient solution, the radioactivity decreased instead of increased as would be expected if the 14 CO2 gas was derived exclusively from biological activity (Levin and Straat 1977), and (ii) no organics were found in the Mars regolith with the Viking GCMS experiment. Although Levin and Straat have responded to these and other criticisms, the biological interpretation of the Viking LR response is considered unverified by most of the exobiological community

(Viking biology experiments: lessons learned and the role of ecology in future Mars life-detection experiments)

[Refers back to 1954 paper by Levin and Strauss, broken link, the text is available here Gulliver–an Experiment for Extraterrestrial Life Detection and Analysis)]

Here the Krebbs cycle also known as the citric-acid cycle is used by eukaryotes as part of the process of using glucose to produce ATP in mitochondria, using up oxygen and producing CO2 as a byproduct (Citric acid cycle). The Emgden path is a simpler faster process that metabolizes glucose to ethanol and CO2 used in yeasts in the process of fermentation and by anaerobic microbes generally - microbes that don't or can't use oxygen. It's also known as anaerobic glycosis (Energetics of Anaerobic Glycolysis)

Clearly this can be done in the orbital laboratory. The iMost team summarize the situation like this

A primary objective of NASA’s Viking Mission to Mars was to search for evidence of extant life.

Two Viking landers carried out a series of biological experiments, including the labeled release experiment, which was designed to detect metabolic activity. In this experiment, simple 14C-labeled substrates, water, and martian soil were combined and monitored for the evolution of radioactive gases (Levin & Straat, 2016) as evidence that microorganisms had metabolized the substrate. Viking instruments detected 14C labeled gas, however, it is widely accepted that those data were due to oxidizing agents in the regolith rather than microbial activity (Lasne et al., 2016).

Despite the controversy surrounding the labeled release experiment and the eventual conclusion that it did not find evidence of life, the rational underpinning of the experiment may be useful in guiding the search for life in the M-2020 returned samples—the idea to search for evidence of active metabolic processes. The orders of magnitude improvements in technology and the availability of sophisticated Earth laboratories would greatly improve sensitivity and allow us to test for metabolic processes beyond the ability to catabolize a handful of simple carbon substrates (a metabolism not likely to exist on the surface of Mars). It would also attenuate the probability of ambiguous and controversial results as seen during the Viking mission.

In this investigation, samples would be incubated with ‘heavy’-isotope-labeled substrate using various combinations of deuterated water, 13C- or 15N-labeled substrates, and other stable isotope labeled substrates (Trembath-Reichert et al., 2017).

(Beaty et al., 2017, iMOST : 92)

This is the method they describe there, using labeled hydrogen and nitrogen as well as carbon - from the source the iMOST team cited:

Taking advantage of the ability to measure three different stable isotope tracers in parallel with NanoSIMS, we applied combinations of ¹³C- and ¹⁵N-labeled substrates in tandem with deuterated water (²H₂O) as a passive tracer for growth. The use of ²H O with NanoSIMS is an effective method for measuring single-cell microbial biosynthesis with high sensitivity, capable of detecting extraordinarily slow rates of growth ... predicted to be characteristic of subseafloor biosphere ecosystems

This multiple-isotope SIP-NanoSIMS study of microbial life in deep subseafloor environments recovered evidence for methyl substrate metabolism in deep coal and shale beds and exceedingly slow growth within this low-abundance, high-temperature microbial community.

Methyl-compound use and slow growth characterize microbial life in 2-km-deep subseafloor coal and shale beds

The chemical explanation of the Viking results is not yet fully worked out
- we need clean samples from Mars to fully replicate the Viking results and make sure we understand them
- which could also use non radioactive isotopes of nitrogen and sulfur to look for other evolved gases

The chemical explanation is not fully worked out yet for the labeled release experiments. Quinn et al. in 2013 suggested that the perchlorates in the soil were decomposed through gamma radiation to hypochlorite (ClO -), trapped oxygen, and chlorine dioxide. Then the hypochlorite reacted with the 14C-labelled alanine to produce chloroalanine which then decomposed to produce the 14C-labelled CO ₂ . ( Perchlorate radiolysis on Mars and the origin of Martian soil reactivity). This didn’t explain everything and a follow up paper by Georgiou et al. filled out the picture some more but is still not a complete explanation (Radiation-driven formation of reactive oxygen species in oxychlorine-containing Mars surface analogues.)

There are points in favour of the hypothesis of life too. Levin and Straat in a paper published in 2016 review some of the issues they have found with this and other abiotic proposals (The case for extant life on Mars and its possible detection by the Viking labeled release experiment.)

• Two of the labeled release experiments got inactivated after storage in darkness for several months
• Activity of the soil is significantly reduced if heated first to 50 °C.

Another interesting sidelight here is Miller ’s reanalysis of the old Viking data in 2002 found an offset of the evolved gases from the diurnal maximum temperature by two hours. This is especially hard to explain by abiotic processes, as the evolved gases would take only 20 minutes to reach the detector. As an expert on circadian rhythms, Miller said they look like circadian rhythms ( Periodic analysis of the Viking lander Labeled Release experiment). He suggested this may be a biosignature in the data. A later complexity analysis seemed to support this interpretation (Complexity analysis of the Viking labeled release experiments). This offset doesn't seem to be discussed further in the literature.

The majority view certainly is that Viking didn't detect life. However it doesn't yet appear to be a complete consensus. We just don't have enough information to reach 100% certainty enough to convince everyone. But this doesn't matter for the experiments. We do the same experiments whatever ones view on the Viking experiments. We can't yet decide for a certainty between:

• it detected life [minority view, majority think this is false,
• there was life there that Viking didn't detect,
• it detected complex chemistry that we don't yet understand fully

This is something we can in principle resolve. That then can be a basis for future studies on Mars. The iMOST team are agreed that we need to do these experiments.

The summary for the 2019 conference on Extant life What's Next suggest using advanced foms of the Viking instruments with the nitrogen and sulfur also labelled (with non radioactive isotopes in those cases).

The results of the Viking biology experiments were equivocal—some aspects of the results were consistent with what would be expected if extant life were present in the samples, but other results were not, thus allowing for multiple interpretations.

Subsequently, scientists proposed explanations for the Viking biology experiment results that do not require the presence of life.

Another type of biosignature of familiar life is the presence of an active metabolism through direct detection of known metabolites such as adenosine triphosphate (ATP).

Metabolic activity of familiar life could be determined by measuring trace gases such as methane and complex volatile organic compounds (VOCs) respired or consumed by extant life.

The uptake of labeled isotopes (C, N, S) such as in the Viking Labeled Release experiment can provide data on metabolic activity and the enantiomer ratio (chirality) of the sample.

Finally, it is important to note that the definitiveness of these results would be dependent on our ability to distinguish signals from noise and would depend on individual results within the context of the entirety of the results.

( Mars Extant Life: What's Next? Conference Report : 801 )

We can find isotope ratios by in situ measurements using mass spectrometers as for the Viking, Phoenix and Curiosity measurements of isotope ratios in the Martian atmosphere (Mahaffy et al.., 2013. Abundance and Isotopic Composition of Gases in the Martian Atmosphere from the Curiosity Rover).

What is clear though is that for the best results, least confusion we need clean samples. One of the issues with interpreting the Viking results is that it detected chlorinated organics which were initially dismissed as contaminants from Earth. Were these the result of the reaction of the perchlorates with surface organics during the analysis which involved heating in an oven? From Curiosity results it now seems that they may be indigenous but there may be traces of organics too.

Following the detection of perchlorates, it was questioned whether the chlorinated organic molecules detected with Viking’s TV-GC-MS experiment were oxidation products of organic molecules present in the pyrolyzed samples. More recently, the Curiosity rover of the MSL mission confirmed the presence of oxychlorine compounds in the regolith of Mars and detected traces of indigenous organic molecules. However, the structure of the molecules present in the regolith may have been altered over geological time in the harsh environment of Mars and may also have been modified during their extraction prior to their detection by in situ analysis instruments.

Consequently, a good knowledge of the reactivity of the martian regolith is essential to understand the possible transformations and ultimately the origin of organic molecules.

(Oxidants at the surface of Mars: a review in light of recent exploration results)

For the clearest resolution of questions like this, which involve trace elements of organics in the dirt we need clean samples of the Martian dirt

Visually identical rocks in the same strata are usually interchangeable for geology
- this is far from the case for astrobiology
- we may need to search many rocks that are geologically identical to find samples of direct interest for past life

It’s also important to realize the situation is very different for life and for geology. With many scenarios the chance of detecting past life in samples returned from Mars is very low until we are able to search for it first in situ on Mars (Seeking signs of life on Mars: In situ investigations as prerequisites to a sample return mission) (Carrier et al, 2020, Mars Extant Life: What's Next? Conference Report. (html) : 802). The life needs to be

• deposited, not eroded away, not chemically altered (Biosignature preservation and detection in Mars analog environments : 4.4. Strategies and priorities),
   
• remain there for billions of years (A field guide to finding fossils on Mars) (Habitability, Taphonomy, and Curiosity's Hunt for Organic Carbon)
   
• not have too much organics added from space to make it unrecognizable (Biosignatures on Mars: what, where, and how? Implications for the search for Martian life : 999 - 1000).

If Martian life didn't evolve as far as photosynthesis 3 billion years ago, past life will be harder to find and if present day life doesn't have photosynthesis either it makes it harder to find too

For instance in one plausible scenario early life on Mars hadn’t yet achieved photosynthesis, or it never progressed to photosynthesis even today. If that is the situation, it could take a lot of searching to find past life.

Ancient martian environments may not have remained habitable long enough for photosynthesis to evolve, and a worthy avenue of research would explore how biosignature signals would be expressed and preserved in this different energy regime.

(Biosignature preservation and detection in Mars analog environments : Section 5. Urgent Needs and Future Research)

Our first clean samples from Mars returned without any in situ life detection may well be as ambiguous for astrobiology and lead to as much controversy as the structures in Martian meteorite ALH84001
- but they still will be valuable as the second step in biological exploration of Mars

Also if we do detect features that look like past life, what we return is most likely to remain ambiguous at this stage as for the Martian meteorite ALH 84001

A more likely scenario is that potential signatures of life might be equivocal, ambiguous or close to detection limits. Such samples might be considered lifeless by some workers and not by others.

…

Although Mars sample return and rover missions are strongly focused on finding evidence for life, lifeless samples returned from Mars will yield important constraints on the extent of habitable conditions and whether those environments were inhabited.

The scenarios discussed here show that it is critical to acquire many samples. Multiple samples must be obtained from each locality, from similar (paleo)environments elsewhere across Mars, and from different potentially habitable environments in multiple locations across Mars for the scenarios presented here to be disentangled and the reasons for lifeless samples ascertained.

(Cockell et al., 2017, Lifeless Martian samples and their significance).

Even with clean bonus samples, it is important to present this to the public as just the second step in Carl Sagan's "Vigorous program of unmanned exobiology" after Viking as the first step
- and the first step for searches for past life
- it would take a significant element of luck to find either past or present day life with the bonus samples
- but we are guaranteed to find out much more about surface chemistry

Video: Open letter to NASA - only the second step in a vigorous program of Sagan's unmanned exobiology

Even with clean bonus samples, it is important to present this to the public as just the second step in Carl Sagan's "Vigorous program of unmanned exobiology" after Viking as the first step - it would take a significant element of luck to find either past or present day life with the bonus samples - but we are guaranteed to find out much more about surface chemistry

Even with the bonus samples we should present this as just the second step in Carl Sagan’s “vigorous program of unmanned exobiology:

Carl Sagan: Because of the danger of back-contamination of Earth, I firmly believe that manned landings on Mars should be postponed until the beginning of the next century, after a vigorous program of unmanned Martian exobiology and terrestrial epidemiology.

(Sagan, 1973, The Cosmic Connection – an Extraterrestrial Perspective)

The two Viking landers were the first step. We have sent no life detection missions to Mars since then. We can expect to make some progress in understanding what Viking saw. But we may well end up with as many questions as we resolved.

• The chemical explanation of the Viking results is not yet fully worked out - we need clean samples from Mars to fully replicate the Viking results and make sure we understand them

Mars is far more complex now than we thought when Carl Sagan wrote that. The surface of Mars is similar in size to the land area Earth's continents and islands. And from a microbial perspective it may be similar in complexity to Earth. See:

• ALL four of the currently confirmed or uncertain novel microenvironments found on Mars relevant to native Martian life were surprises
   
• Predictions from the literature for seven potential microenvironments we can't expect to spot from orbit
   
• Scenarios of microbial oases with unique species local to them - Intriguing possibility that transfer may be possible but only for some especially hardy species on rare occasions - analogy of the Polynesian islands - in these scenarios, Mars could have numerous microbial oases each with unique species of microbes that evolved separately for millions of years

So we are likely to need many missions for a reasonably thorough exploration of Mars. We are at great risk of forward contamination if we send many robotic explorers to sensitive areas of Mars using the Perseverance level of cleanliness or even the far more rigorous Viking mission level of cleanliness.

It’s important not to present this to the public as the mission that will settle central questions of astrobiology. With better samples in clean containers, it may detect past or present day life, but it would take a significant element of luck to be so successful with a first mission.

Rather, this is the second step in our exploration of Mars to search for life (the Viking missions would be the first step as no mission since then has tried to find life).

It is also just the first step for the search for past life.

This suggestion of a miniature telerobotic laboratory would help NASA retain your world-leading roll in planetary protection - and if we find life on Mars that can never be returned to Earth like mirror life it would be the core of a collaboration similarly to the ISS in miniature and at much lower cost for participating nations

Video: Open letter to NASA: this suggestion maintains your leading role planetary protection astrobiology

My suggestion is just one solution, which I also mentioned in the final comment on your EIS (Walker, 2022, Comment posted December 20th). In this way you can move forward from this EIS with a new approach that would

• retain your world-leading role in planetary protection,
• be an excellent example that’s easy for other space agencies and private space to follow to keep Earth 100% safe - they can either just sterilize their samples - or they can return to a satellite above GEO like you
• It could be the seed of a growing “miniature space station” but of small modules a few meters across staffed by small telerobotic handlers and miniature instruments rather than humans.
• It could include a very excellent Mars simulation chamber using a centrifuge to simulate Martian gravity with windows that let in appropriately filtered sunlight, simulating a day night cycle inside which could be used for testing terrestrial organisms in Mars surface conditions or reproduce Mars surface conditions to try to get returned life to grow in the satellite.
• likely has less upfront cost this decade - no BSL-4 and this suggestion - and it may not even add to the mass for the Earth Return System - electropropulsion is ideal for minimum energy transfer orbits it needs only a few meters per second delta v for capture by the Earth Moon system, and move to the Earth Sun L2, the Earth Moon L2 and then more delta v to get to its final orbit - but this saves on the aeroshell and the fuel to ship the aeroshell to Mars and back
• adds the cost of a small satellite in a safe orbit above GEO in the 2030s (when launch cost to GEO will likely be greatly reduced)
• with a suggestion for small bonus samples gathered by the ESF rover in a clean container that would greatly enhance the science return for astrobiology.
• Achieves far more science return, especially for astrobiology
• Has far less legal complexity and can be done with a simple NEPA statement similarly to your sample returns from asteroids and comets
• Stimulates universities and other research institutions to work on novel miniature life detection instruments such as SETG to be space certified and send to the orbital telerobotic lab - which will then be exceptionally useful for in situ exploration on Mars - these instruments are also likely to have many terrestrial applications
• Prepares the way for future inspiring missions to Mars
  Also of great value for future astrobiological exploration of other locations in the solar system
  - such as to study samples returned from Ceres, Europa's ocean, Enceladus, Titan and so on

What other suggestions might you get if you open up communications to other scientists and the general public in an open ended way? There may be many solutions just as there were many ways the Titanic could have avoided the iceberg - this suggestion just shows there is at least one way to do it

What other suggestions might you get if you open up dialog with the wider community of other agencies, international organizations and scientists in other countries, and the general public?

There may be many ways to do this, just as there were many ways the Titanic could have steered to avoid the iceberg - but the sooner this happens, the better, for NASA, for planetary exploration science, for astrobiology and for the public.

The best way to follow up from this mission to complete Sagan's "vigorous program of unmanned exobiology" is to use dozens or hundreds of 100% sterile landers and rovers on Mars
- and we have the technology now to do it with HOTTech [IN NEPA BOTH]

This is just the second step of what's likely to be many steps to get an understanding of any Martian biosphere or find out if it has present day life or had life in the past, see above:

• Even with clean bonus samples, it is important to present this to the public as just the second step in Carl Sagan's "Vigorous program of unmanned exobiology" after Viking as the first step
  - and the first step for searches for past life
  - it would take a significant element of luck to find either past or present day life with the bonus samples
  - but we are guaranteed to find out much more about surface chemistry

It will be a huge plus for the next phase of Mars exploration if we can have 100% sterile rovers.

We can do that with some help from NASA's (NASA, n.d., HOTTech) technology, a program to build a Venus surface landers that can survive and function for at least 60 days at 500°C without any cooling.

"The goal is to develop technology areas that will enable a long-lived lander that can survive at least 60 days at 500C." (NASA, n.d., HOTTech)

We can already build a complete surfacing surface probe on Mars that can not only be heated to 500°C without damage for months on end even function and do its job observing Mars (Long-lived in-Situ solar system explorer (LLISSE) Potential Contributions to solar system Exploration)..

Here are some of the components in development, to work at 500°C without damage for anything from days to months on end (HOTTech)

• electric motor and position sensor
• UV near field imaging (beyond the optical resolution limit of normal microscopes)
• radio transmitter
• solar arrays (to last at least a day at 500°C)
• batteries
• ... for more see (HOTTech)

However 500°C is higher than we need to sterilize a rover 100%. We just need to be able to heat it to 300°C for a few minutes on the journey out to Mars to send 100% sterile rovers to Mars. See below:

• The specification for a Marscopter that can be heat sterilized before it lands on Mars is far simpler than for a Venus probe
  - components need to function at normal temperatures after brief heating for a few minutes at 300°C and don't even need to function at 300°C never mind 500°C

That opens up many more possibilities that we can use right away.

• We have high temperature components commercially available for use for long periods above 200°C, developed for electric aircraft (where they save weight placed close to the engines), electric cars, and oil wells amongst other uses. This paper is from 2012 and technology has advanced since then but it outlines the field (High-temperature electronics pose design and reliability challenges). It also mentions the idea of testing components above their design temperature - especially since we just need them to resist a few minutes at 300°C we may find many components can do that already
• Fast electronics operating at 300°C with 0.35 micron resolution (High temperature electronics) (Integrated High Temperature Circuits - Fraunhofer IMS) using silicon in insulator technology (Silicon On Insulator (SOI))

Pioneer - the 2018 largely mechanical automaton rover for Venus and Wilcox's 2017 design for a 100% sterile cryobot for Europa's ocean that can be heated to 500°C for sterilization

Video: NASA Engineers Design an Indestructible Venus Rover

The original idea was a wind powered almost fully mechanical rover on Mars.

The researchers proposed (in 2018) that the same approach could be useful for planetary protection because of their rover's resilience to heat sterilization, with an automaton rover explorer deployed from a more capable but less fully sterilized master rover to visit a nearby sensitive habitat (Automation Rover for Extreme Environments : Section 6.2).

In 2017, Wilcox designed a Europa probe that would be heated to 500°C on its journey out to Europa for complete sterilization (A deep subsurface ice probe for Europa)

These pioneering ideas have become much easier as a result of advances in technology for HOTTech in just the last 5 years. We now have the capability to build a fully functioning probe that can survive for months on the Venus surface at 500°C with the components needed for a science mission (Long-lived in-Situ solar system explorer (LLISSE) Potential Contributions to solar system Exploration).

The specification for a Marscopter that can be heat sterilized before it lands on Mars is far simpler than for a Venus probe - components need to function at normal temperatures after brief heating for a few minutes at 300°C and don't even need to function at 300°C never mind 500°C

I suggest we start with sterile Marscopters which would let our rovers send the marscopters to nearby features that they are not sufficiently sterilized to explore themselves and work up to a future where all missions to Mars are 100% sterile. This builds on a suggestion from the Venus lander team from 2018 which originally was almost fully mechanical. - it becomes far easier already to have 100% sterile probes. See above:

• Even with clean bonus samples, it is important to present this to the public as an important second step in Carl Sagan's "Vigorous program of unmanned exobiology" after Viking as the first step
  - and the first step for searches for past life
  - it would take a significant element of luck to find either past or present day life in the bonus samples
  - but with bonus samples we are guaranteed to find out much more about the surface chemistry of Mars
  - preparing for the next steps in a potentially very rapid robotic exploration of Mars

With

• so much technology development in the last five years and with
• much lower specs, just to survive for a few minutes at 300°C

At 250 °C the half life of the RNA bases under hydrolysis is between 1 and 35 minutes, and at 350 °C the half-lives are between 2 and 15 seconds (The stability of the RNA bases: implications for the origin of life : 7935)
 

• Eight of the 20 amino acids have been proven to not just evaporate or liquify but to decompose at temperatures between 185°C for Q (Glutamine) to 280°C for H (Histamine) Thermal decomposition of the amino acids glycine, cysteine, aspartic acid, asparagine, glutamic acid, glutamine, arginine and histidine)
   
• There would be other more recalcitrant organics remaining but a few minutes at 300°C would already be enough to eliminate viable terrestrial life and may be enough to eliminate the most problematic chemicals for giving “new ideas” to prebiotic chemistry.

Perhaps we could already build a Marscopter that could be sterilized to 300°C as it has relatively few components compared to Perseverance. Most of them seem to exist already in the (NASA, n.d., HOTTech) program and many others such as video cameras already exist in commercial use, for instance to look inside ovens, and could resist a few minutes at 300°C easily.

In summary, we are now able to protect Earth 100% in the backwards direction with almost all instruments needed for the iMost experiments - with gene sequencers to complete the picture by 2033 and have a realistic possibility of 100% sterile rovers on Mars by the 2030s [IN NEPA BOTH]

Video: Open letter to NASA - how we can explore Mars for life rapidly with broadband and telerobotics

Text on graphic: 100% planetary protection both ways, even if Mars has vulnerable early life, prebiotic chemistry, or mirror life.

Using NASA’s remarkable Venus HOTTECH technology, all these surface assets can be built to withstand months at 500°C, and so are easily sterilized with a few minutes of heating on the journey out.

Main image: “Safely tucked inside orbiting habitat, space explorers use telepresence to operate machinery on Mars, even lobbing a sample of the Red Planet to the outpost for detailed study." (Telerobotics Could Help Humanity Explore Space)

This is our choice as a civilization - I hope you agree that this is an interesting vision and one that is worth looking at carefully and proposing to the public as an alternative to dropping planetary protection

I conclude we have technology already to achieve 100% protection of Earth, and we have technology in development which could be accelerated to protect the rest of the solar system from terrestrial contamination, enabling 100% planetary protection in both directions, and we can do this with no loss of science return. Indeed by opening up the most sensitive areas of Mars, Europa’s ocean and so on to 100% sterile robotic explorers, this new technology has potential lead to a huge advances in science.

It is our choice as a civilization whether we do this.

We don't know which scenario we face - a Mars where humans can SAFELY explore the surface - or one where they can NEVER explore the surface safely or one where we need PRECAUTIONS such as quarantine to do it safely

I hope you agree with this, and we can work together to find a way forward to a stimulating future. We don’t know which of these scenarios we face, a future where it is:

• SAFE for humans to explore the surface of Mars in person with no precautions needed in either direction
  (such as no life or mutually benign life on Mars)
   
• WITH LIMITED QUARANTINE and other measures for travel between the planets to keep out certain invasive species that would harm Earth’s biosphere, humans, or agriculture.
   
• NEVER SAFE for humans to explore Mars in person - or at least, never return to Earth after doing so
  (such as life based on mirror organics on Mars)

That is why we need to do planetary protection. We need an open mind here, to look at this with clear eyes and develop a Mars exploration program that is inspiring and stimulating for both robotic and human missions no matter which scenario we face.

Mars is the only terrestrial analogue of Earth within light years
- if it has interesting prebiotic or abiotic chemistry
- such as flipping chiral networks
- terrestrial contamination would make it impossible to study this in action
- the order we introduce microbes might also matter for colonization

Video: Open letter to NASA: even prebiotic chemistry on Mars could be of extraordinary interest

For Mars to be like the Moon, with no issues for humans visiting there in the forward direction, we need Mars to have:

• no interesting prebiotic or abiotic chemistry - Mars is the only terrestrial analogue of Earth within light years - once we introduce terrestrial life no future generations on Earth will ever be able to study current chemical processes on present day Mars, just hints of how they worked in protected deposits.
   
• where it doesn’t matter what order terrestrial microbes are introduced to Mars - some terrestrial microbes may be able to turn subsurface aquifers on Mars to cement for instance.

Mars could have uninhabited habitats (Cockell, 2014, Trajectories of Martian habitability). If Mars has interesting prebiotic or abiotic chemistry that would be erased by terrestrial life, and we introduce terrestrial life to start the anthropocene on Mars, we become the last generation with the opportunity to study prebiotic or abiotic chemistry on a terrestrial planet in our solar system. This would seem to be a decision that needs a broad consensus in our civilization given the wide-ranging effects for future generations of our actions.

We can be pretty sure there are viable terrestrial; microbes on Mars. attached to our spacecraft and some have likely fallen into the dirt - but that's not the same as starting the anthropocene on Mars, because they also need to find habitats that they are pre-adapted to inhabit, which most likely hasn't happened yet. Also if we do find terrestrial life has started to spread irreversibly, this doesn't mean that it will make no difference to introduce extra species. Cane toads have different effects on Australian wildlife from European rabbits. Methanogens would have different effects from photosynthetic life and so on.

We need a reality check on the timescale - it is easy to send dead humans to Mars but we don't yet have the technology for a self sustaining base on the Moon that can operate without resupply from Earth for several years

We need to be realistic about the timescale for humans to Mars orbit - if we had that technology already we could set up a permanent base on the Moon with far less expense than for the ISS. NASA want to build a permanent base on the Moon. If we had the capability to send humans to Mars, we could send, say, a half dozen astronauts to the Moon with all the supplies they need for 3 years, and then leave them exploring and never need to send more supplies, replacement parts etc. This would make it very easy to establish a permanent base on the Moon. It would likely cost far less than the ISS. But we are nowhere near that level of technological readiness.

We couldn’t send an ISS clone to Mars, not safely. The ISS needs resupply every few months and they frequently need replacement parts from Earth when components fail.

The ISS crew also have lifeboat spacecraft attached to the station. Though it’s never been needed, they can get back to Earth in a few hours if they have a fire, chemical release, explosion, or a micrometeorite that damages the ISS to the extent they can’t repair it and need to evacuate right away. When we explore Mars it will be a two day medvac back to Earth. For missions to the Moon it’s nearly two years medvac if there is an accident just as the crew are leaving Earth on their way to Mars.

The Moon is hard to get to and far more interesting than we realized in the 1960s and 1970s - many adventures there before we go further
- Chris Hadfield
- former commander of the ISS thinks ultimately we will be living on the Moon for a generation before we go to Mars
- "It’s as if you and I were in Paris, paddling around in the Seine in little canoes saying, 'We’ve got boats, we’ve got paddles, let’s go to Australia!' Australia? We can barely cross the English Channel."
[IN NEPA BOTH]"

The Apollo astronauts made it look easy - but that was because they were experienced test pilots who could make quick decisions in an emergency without even a moment of panic - like when the Apollo 10 astronauts got within a few tens of seconds of crashing into the Moon (Teitel, When Apollo 10 Nearly Crashed Into the Moon) or when Buzz Aldrin found a way to take off from the lunar surface using a felt tip pen (Tell Me A Story: Buzz Aldrin's Felt Tip Pen Saves Day)

The retired Canadian astronaut Chris Hadfield, former commander of the ISS, interviewed by New Scientist, put it like this:

"I think ultimately we’ll be living on the moon for a generation before we get to Mars. If the world and the moon were threatened and the only way to preserve our species was to launch from Earth, we could go to Mars with yesterday’s technology, but we would probably kill just about everybody on the way."

(Klein, 2017, (Chris Hadfield: We should live on the moon before a trip to Mars)

It’s as if you and I were in Paris, paddling around in the Seine in little canoes saying, “We’ve got boats, we’ve got paddles, let’s go to Australia!” Australia? We can barely cross the English Channel.

Frame from 28 seconds into this ESA video: Moon Village

The Moon is a place where we can make our first steps in sustainable living in space, within easy access of Earth for repairs, supplies, and emergency medvac back to Earth in only two days. It's far more interesting than we realized in the 1960s and 1970s.

The lunar caves are truly vast far larger than lava tube caves on Earth. Some may be up to kilometers wide. Some of the lunar caves probably have an internal steady temperature of around -20 °C, potentially useful as a constant heat sink for a settlement (. Lunar and martian lava tube exploration as part of an overall scientific survey) The challenge of providing energy during the lunar night is a similar challenge to providing energy during Martian dust storms. Then there are the peaks of almost eternal light at the poles with solar power 24/7 nearly year round (Peaks of Eternal Light), the polar ice and so on (, Moon’s South Pole in NASA’s Landing Sites).

The Moon is also far easier to develop commercially in the near future. We may have hundreds or thousands on the Moon before we are ready to make that big and difficult step onwards to Mars in a reasonably safe way if that's the decision.

If we have a mirror life terrestrial planet in our solar system - this will surely lead to more not less interest in space exploration and settlement - we'd need to travel light years to find another one

If we do find life on Mars based on an independent biology, it's a discovery to treasure, not a reason to despair.Carl Sagan called it a "treasure beyond assessing". Mirror life would fit this.

The existence of an independent biology on a nearby planet is a treasure beyond assessing, and the preservation of that life must, I think, supersede any other possible use of Mars.

(Happy Birthday Carl Sagan)

Space colonization enthusiasts might feel their dreams are shattered. But far from it. We can see this as an inspiration for space exploration. To have something so remarkable, in our own solar system, which we'd otherwise likely need to travel light years to find, especially independent life on a terrestrial planet (we might also find independent life in the oceans of the icy moons Europa, Enceladus, and the asteroid Ceres amongst others, but it would still be of unique interest to find independently evolved life in a terrestrial world like Earth).

We have places on Earth we can’t settle currently such as the deep ocean, even the shallow ocean is hard for us to inhabit, for now the atmosphere is hard to inhabit, we have only made a start on sea steading, and some places are well beyond current technology. We can’t explore down to the core of our planet as Jules Verne envisioned in his “Journey to the center of the Earth” (A Journey to the Center of the Earth). But we have never had a frontier due to biology.

A Mars with mirror life, perhaps, becomes a forever unattainable frontier, a world you can never actually land on, but can still explore with high fidelity telepresence. We have never had a frontier like that.

If we do find life on Mars that can never be returned safely, this may stimulate rather than discourage vigorous space exploration and settlement. The first astronauts to Mars might study the surface remotely in a spectacular orbit, a sun synchronous Molniya orbit as proposed by the Mars HERRO study. This orbit is tilted at 117 degrees and it is easy to get to as it needs less delta v than a landing on Earth’s moon, and is similar to the minimal delta v Mars capture orbit

• The spectacular HERRO orbit for our first humans in Mars orbit controlling assets on the ground including telerobotic avatars
• Next step as our spacecraft get more capable - Jupiter's moon Callisto - far better for humans than Europa, naturally shielded from ionizing radiation and no planetary protection issues either way as it is classified similarly to the Moon for planetary protection
• Then Titan - the only Moon with an atmosphere, 1.5 times Earth's atmospheric pressure - - far easier to protect against cold than vacuum - probably no planetary protection issues either way if it doesn't have cryovolcanism - so exceptionally cold any life there is unlikely to be able to survive on Earth
• Finally with space habitats slowly spinning for artificial gravity and large thin film mirrors we can colonize out to Pluto and beyond

 

 

If Space Colonization enthusiasts are right they will have numerous assets on the surface by the time we are able to confirm their intuition that it is safe to colonize Mars and if we can't colonize Mars it's not such a big step to go to the asteroid belt or Cailisto or Titan as with an evac time of months to years then there is no way to deal with an emergency by returning to Eart

If space colonization enthusiasts are right that the two biospheres are compatible, by the time humans get to Mars they will have numerous assets on the surface of Mars already.

This compares evacuation times:

ISS emergency evacuation a few hours, resupply every few months < day to arrive

Moon emergency evacuation 2 days, resupply takes 2 days to reach the Moon

Mars emergency evacuation minimum 6 months, emergency resupply minimum 6 months to arrive

(added text to this infographic from the Canadian space agency: Distances between Earth and the International Space Station, the Moon and Mars - infographic)

Actually especially with faster rockets, there's a much bigger difference between traveling to the Moon and traveling to Mars, than there is between traveling to Mars or traveling to Jupiter's moon Callisto. Because we need superreliable life support that can last for years without resupply for either and capability to deal with medical emergencies with essentially no possibility of medvac ito deal with any emergency health issue.

Broadband communications with Mars will make a huge difference
- it would be like turning NASA's HiRISE satellite which can take photos from orbit with a resolution of less than a meter into 18 satellites with the same capability
- and our rovers would be like several rovers each
- we currently operate them in the same way we would operate a rover as far away as Pluto!

At present the bandwidth is so low that even with a round trip latency which at its best is less than 7 minutes, Perseverance and Curiosity’s teams can only download data from the previous day which they then use to plan operations for the next day which they then send up in commands to Mars. It would be as easy to control a rover on Pluto with round trip latency of 11 hours as a rover on Mars! (NASA Mars Perseverance: A Sol in the Life of a Rover)

[In 2003, the distance was 55,758,006 km center to center (Mars Close Approaches Archives - NASA), or 186 light seconds (55758006 km in light seconds), round trip latency 6 minutes and 52 seconds]

We will need a huge increase in bandwidth from Mars once we have astronauts in orbit there anyway. This will make an ENORMOUS difference to Mars exploration science.

HiRISE is the remarkable high resolution optical telescope orbiting Mars which achieves sub-meter resolution photographs of the surface. However, it is limited in the number of photographs it can take of Mars mainly because of the bandwidth. It can only send back 6 Megabytes per second.

At MRO's maximum data rate of 6 megabits per second (Mbps) (the highest of any Mars mission), it takes nearly 7.5 hours to empty its on-board recorder and 1.5 hours to transfer a single image back to Earth that the onboard High Resolution Imaging Science Experiment (HiRISE) camera has taken. In contrast, with an optical communications solution at 100 Mbps, the recorder could be emptied in 26 minutes, and an image could be transferred to the Earth in less than 5 minutes.

(Benefits of Optical Communications )

So at 100 Mbps with optical communications, it will be able to return 18 images in the time it currently takes to return one image.

At MRO's maximum data rate of 6 megabits per second (Mbps) (the highest of any Mars mission), it takes nearly 7.5 hours to empty its on-board recorder and 1.5 hours to transfer a single image back to Earth that the onboard High Resolution Imaging Science Experiment (HiRISE) camera has taken. In contrast, with an optical communications solution at 100 Mbps, the recorder could be emptied in 26 minutes, and an image could be transferred to the Earth in less than 5 minutes.

(Benefits of Optical Communications )

So it will be able to return 18 images in the time it currently takes to return one image with optical communications.

(Deep Space Optical Communications (DSOC))

There is no way we continue at this low bandwidth once humans are in orbit around Mars. We can put this in place long before then. And NASA are listening, they plan to send a technology demo on the Psyche mission when it launches to visit the asteroid of the same name - a huge asteroid made entirely of metal. The Deep Space Optical Communicator will be tested as far as its flyby of Mars and possibly for longer in an extended mission (Deep Space Optical Communications (DSOC) ) Psyche will launch in 2023.

Meanwhile using artificial real time from computer games and fast bandwidth communications from Mars we can explore Mars from our own homes on Earth as if we were there
- even look at rocks with a hand lens based on a virtual 3D world built up from gigapixel streaming video from Mars

Meanwhile, we can speed the Mars exploration so much that using this idea of "artificial real time" from computer games, we can control our rovers almost as easily as a rover on the Moon (say), which NASA will be able to do once you have broadband optical laser communication with Mars later this decade.

It works by simulating the position of your rover as it will be when the commands get to Mars, so you can drive in a virtual landscape in real time with no latency, a technique used in online multiple player games. In this video he talks about using way points - so you tell the rover where you want it to go, and so long as the waypoints are ahead of where it has got to it doesn’t slow down.

Video: Telexploration: How video game technologies can take NASA to the next level

This was later developed into OnSight using Microsoft’s “hololens” software.

Video: Walking on Mars w/ HoloLens [OnSight]

For more about it: (Immersed on Mars — Dr. Jeff Norris)

It will help also to have more autonomous and more rugged rovers.

By the 2030s with fast bandwidth and once we have gigapixel video cameras on Mars we’ll have scenes like this, but far higher resolution than this, building up a 3D virtual world in microscopic detail as the rovers traverse Mars.

(First 4.5-billion-pixel of Mars by NASA's Perseverance Rover)

With gigapixel video cameras on the rovers, streaming back broadband to Earth, scientists and enthusiasts will be able to look at the landscape on Mars with a virtual hand lens, examining any rock the rovers ever passed by in microscopic detail. Their discoveries could then lead to the rover turning around and looking at the rock again - but as rovers get faster, able to move tens of kilometers a day as for the lunar rovers or even faster - and not limited like the lunar rovers were, to remain within walking distance of a base (The Apollo Lunar Roving Vehicle). We need far faster and more robust rovers for human exploration if we do get humans on the surface. We can send them there first, for robotic exploration.

For astronauts in orbit around Mars it would be not unlike playing a game of "civilization" with many semiautonomous rovers on the surface, driving by themselves with assistance from Earth already

In this way we would have many assets already in place when the astronauts get there, and data links working and tested, and capable teams on Earth to do most of the heavy work leaving it for the astronauts in orbit to be used to their maximum for the executive capabilities. For them it might be not unlike playing a game of “civilization” stepping in from time to time to help one of the robots that is stuck on some task or needs to be operated directly for some time sensitive experiment.

So we can do a huge amount of exploration on Mars from Earth in the very near future, and all this with those near future 100% sterile rovers that can explore anywhere with no planetary protection restrictions. All of this builds up assets on Mars and knowledge about Mars that will be very useful when humans get there, whether in orbit or on the surface.

If the colonization enthusiasts are right they get a "pass" and have much broader backing because the rest of our civilization knows they are right
- hardly interrupting their plans- and if they have to stay in orbit
- they can use those assets to exploit Mars and even set up robotic farms on the surface which would work even on a mirror life planet because seeds can be sterilized unlike humans

The Mars colonization enthusiasts are so sure that the Martian biosphere will be safe for Earth. If they are right we will get a “pass” as a result of this exploration. It will hardly interrupt their plans at all if their confidence is well placed. Not only that they would get much broader backing because their confidence will be based on scientific knowledge about Mars rather than optimistic hunches and vivid metaphors.

While if they have to stay in orbit, and colonize the moons of Mars, they can use these assets to exploit Mars with robotic exports to their colony, even grow plants on the surface.

Though we can’t sterilize humans, we can sterilize seeds without any risk of contaminating Mars with Earth micro-organisms.

Text on graphics: We could grow plants on Mars even if it has mirror life that can never be brought back or Earth has microbes can never be sent to Mars.

Seeds can be sterilized and grown in sterile aquaponics

(The Real Martian Technologies: Our Little Green Friends)

In this way we could have greenhouses on the surface of Mars and these could grow food for the colonists in orbit. They may have plenty to eat in their habitats anyway by then, but they might grow maybe delicacies or things that grow particularly well on Mars or medicinal plants or whatever. Also they might grow large plants, and maybe trees (perhaps growing far larger in the Mars light gravity) or whatever else grows best on the surface of Mars, or for convenience to save space in orbiting habitats.

We can in principle grow many terrestrial crops on the surface of Mars with no risk of contamination in either direction, on most scenarios. We could export those crops to the orbiting colonies, for instance on Phobos. We can also do mining for minerals on Mars, and prospect for assets that may be worth selling to Earth and so on, do everything the colonization enthusiasts want to do except humans on the surface, much like the game of civilization with robotic avatars.

I believe the general public needs to be aware we have this choice as a civilization
- and the decision needs to be made on a broader basis than internal technical discussion within NASA
- to find a way forward that is better for NASA, for the general public, for planetary science, for your reputation
- and good for space colonization enthusiasts too whatever their views on the need for planetary protection

I believe the general public needs to be aware 100% protection is available and is scientifically feasible in both directions. This decision needs to be made on a broader basis than internal technical discussions within NASA. So it’s important to open up discussion and engage more generally with other agencies, your former planetary protection officers, and the general public. More generally it seems clear you need to move in the direction of more not less communication with others as you work on the project.

By sending this open letter I hope to encourage you to participate in a wider discussion with experts of many disciplines and the general public in order to find a way forward that is good for NASA, for the general public, for planetary science, and for your reputation. Also to find a way forward that is good for the space colonization enthusiasts too whatever their views on the need to protect Earth's biosphere. I am presenting here some of the things you will need to listen to in the future. I hope this will lead you to a more open direction.

With the smoke detector analogy this is my proposal.

Hand installing smoke detector labeled “NASA” and wooden ceiling of a house labeled“Earth”

Text on graphic: Actually we can do better than a smoke detector.
- 100% protection of Earth
- Costs less than NASA's plan
- Far more science return

(Smoke detector graphic from The EnergySmart Academy)

[I’ll archive this open letter with a do in a preprint server for future reference before sending it as for my preprint - url to preprint here]

---

SUMMARY OF THE 14 MAIN POINTS AS PRESENTED IN MY PUBLIC COMMENTS UNDER NEPA

Let’s look at a summary of the main points. These were presented on the last day of public comments to your draft EIS. Since then my preprint and literature survey added many more details but hasn't led to any changes in those main points.

I cover the same points in the first round, some of them in an attachment in the first round but the reasonable alternative and the issues with complying with the ESF recommendations were stated clearly in the comment itself in the first round too.

First as presented on 20th December:

I recommend this draft Environmental Impact Statement is stopped, and a new one prepared after doing the necessary size limits review, and fixing whatever led to its many errors.

1. The BSL-4 recommendation in this EIS is out of date, based on science of 1999.
2. This EIS does not mention the most recent Mars Sample Return study from 2012 by the European Space Foundation which reduced the 1999 size limit from 0.2 microns to 0.05 microns to contain ultramicrobacteria and required 100% containment at that size.
3. A BSL-4 is not designed to this standard. In recent reviews of filter technology, I find NO AIR FILTERS with that capability – and no evidence anybody is working on them. Air filters for larger particles remove some of these very small particles kicked out of the airstream by jostling of air molecules by Brownian motion but can't remove all. It is an unusual requirement.
4. NASA haven't responded to my comment in May which alerted them to this omission. They still don't cite the ESF study. Also, the ESF said their limit needs to be updated periodically. An update is certainly due a decade later.
5. The EIS has an overnarrow scope in the Purpose and Need section - it requires samples to be returned unsterilized to terrestrial labs for "safety testing". This won’t work. NASA believe they reduced the most abundant biosignatures to 0.7 nanograms per gram of returned rock sample – this guarantees a positive test. There will be no way to know if tubes contain safe terrestrial life or potentially unsafe martian life.
6. This narrow scope improperly excludes the reasonable alternative of presterilizing samples before they reach Earth's biosphere - which achieves virtually the same science return and keeps Earth 100% safe. By a 1997 case in the 7th circuit this alone probably invalidates the EIS.
7. The high levels of forward contamination make astrobiology almost impossible. I recommend bonus samples of dirt, dust and atmosphere collected in a STERILE container with no terrestrial organics, brought to Mars, especially on the ESA fetch rover.
8. I recommend returning these bonus astrobiology samples to a safe orbit above GEO where they can be tested for life
9. The EIS’s reasoning for no significant environmental effects contradicts the conclusion of the NRC study from 2009 which they do cite, which says the risk of even large-scale impacts on human health or environment is likely low but not demonstrably non zero. It also warns against the meteorite argument that they use. I found multiple errors in my analysis.
10. Returned life COULD be harmful. Example, fungi kill crops, other life and sometimes immunocompromised humans. Botulism, ergot disease, tetanus, all are the results of exotoxins not adapted to the lifeforms they kill, similarly some algal blooms kill dogs and cows that eat them. BMAA misincorporated for L-serine causes protein misfolding and is a neurotoxin implicated in some cases of the disease that affected Steven Hawking - an alternative biochemistry may have many different amino acids similar enough to terrestrial amino acids to be misincorporated. Or perhaps martian life evolved from scratch from mirror chemicals as mirror life - the effect on our biosphere can't be predicted. I give many such examples in my preprint. Or it could be harmless like microbes from a terrestrial desert, or indeed beneficial. But we DON'T KNOW. So we need to find out first.
11. What matters for invasive species are the ones that can’t ‘get here, like starlings that can't cross the Atlantic rather than barn swallows. The freshwater diatom Didymo is invasive in New Zealand and can't get from one freshwater lake to another without humans. A microbe adapted to briny seeps on Mars and to spreading in dust storms shielded from UV, may well not get to Earth in a meteorite, while a sealed sample tube including Martian atmosphere, at Mars atmospheric pressure, is like a mini spaceship.
12. Quarantine of humans can’t keep out a fungal disease of crops, mirror life etc.
13. So any unsterilized samples will need to be studied remotely via telerobotics which also greatly reduces forwards contamination (issues with filtering ultramicrobacteria will go both ways).
14. Astrobiologists now have tiny instruments that can go from sample preparation to life detection, even to a gene sequence[r], operated remotely on Mars. They could send hundreds of these in each 7 ton payload of the Ariane 5 to above GEO.

Let's make this an even better mission and SAFE for Earth.
Thanks!

(Walker, 2022, Comment posted December 20th)

The 8 attachments to the 20th December comment go into this in more detail

I cover many other points in the attachments, two of which I also have as preprints on the OSF server. They are written to high academic standards but I don't intend to submit them for publication as this is not needed for the purposes of the EIS and would take time.

Since we can't link directly to the files online on the NEPA website I link to my own copies of the files exactly as they were on the day I uploaded those attachments which you can find in my "NASA_MSR_comment-as-sent" folder.

From an academic point of view, the main attachments to (Comment by Robert Walker posted December 20th) are:

• (Walker, 2022, So many serious mistakes in NASA's Mars Samples Environmental Impact Statement it needs a clean restart) (docx)
  - A detailed analysis of the mistakes in some of the main planetary protection statements in the EIS
  with cites and with headers of sections as mini-abstracts so you get a good first idea by reading just the top level headers.
  [Online copy of the exact file I sent to NASA for the final comment and the word document I used to export the pdf as it was on the day when I exported the pdf]
  
  [I did this to a deadline and there are a few sentences in the EIS that I missed in this analysis that I cover in this open letter labelled as [NEW] - also I had no idea of the recent history of NASA that led to the numerous mistakes - just analysed what I saw in the draft EIS]
  
  I did some minor updates after the EIS was over. The latest preprint is here: (Walker, 2023, So many serious mistakes in NASA's Mars Samples Environmental Impact Statement it needs a clean restart - omits major impacts – uses old science later overturned – statements cited to sources that say the opposite – no response to significant public concerns – “Purpose and need” should permit a sterilized return as a reasonable alternative action - and size limit needs a new review first, a decade after the ESF in 2012 reduced the size of particle to contain from 0.2 to 0.05 microns)

and

• (Walker, 2022, NASA and ESA are likely to be legally required to sterilize Mars samples to protect the environment until proven safe) (docx)
  [this is the same file as the preprint of similar name from the first round of public comments updated for instance to take account of the fast track NEPA process - and the word document I used to generate it as it was on the day I exported it]
  Again with headers of sections as mini-abstracts so you get a good first idea by reading just the top level headers.
  
  This is about NASA's mission generally and not specifically about the EIS - but it has extensive discussion of issues for planetary protection for NASA's mission such as lab leaks and quarantine, and numerous planetary protection scenarios to consider for alien life including mirror life, fungi, and many ways that microbes can be harmful other than direct infection of humans, and covers issues with transport of life in dust storms, issues with terrestrial contamination of the samples, and so on - which is why I added it to the open letter.
  
  I continue to fix minor errors, add cites and so on , so if interested, the latest preprint is here: (Walker, 2023, NASA and ESA are likely to be legally required to sterilize Mars samples to protect the environment until proven safe)

I used a lot of material from those two attachments as the basis for my new preprint and literature survey (Walker, 2023, NASA must protect Earth's biosphere even if Mars samples hold mirror life) , and I use that in turn as the basis for this open letter. To help with clarity I have added [NEW] to sections in this page that are based on material new since my public comment. It is important to consider those issues too, however the open letter focuses on the ones that I have legal standing for under NEPA as I raised them as issues in a timely fashion during the public comments period.

Then there's an attachment that gives some background about me as recommended

• About me

The other attachments in the final comment are pdf versions of blog post I did about the issues I found - for the most part they cover material already covered in those more academic posts in a less technical way.

• Why doesn't NASA respond to public concerns on its samples from Mars environmental impact statement
  [N.B. the links to my YouTube video on the topic and other links in that attachment don't work for some reason - I didn't notice until much later
  - but it is a copy of this blog post (which has some minor fixes) with the links working correctly
  : NASA - Do Listen To Public Concerns About Life In Samples From Mars - Your Plan Is Like Building Us A House With No Smoke Alarms]
   
• NASA - Your Samples From Mars Need A Better Than Biosafety Level 4 Facility - NOT Designed To Contain Even Earth's Tiniest Cell
   
• Dear Space Explorers - Yes We Do Need To Protect Ourselves And Earth From Any Microbes In Mars Rocks As We Explore
   
• So many serious mistakes in NASA_s Mars Samples Environmental Impact Statement it needs a clean restart
   
• NASA and ESA are likely to be legally required to sterilize Mars samples to protect the environment until proven safe
  

Then there's the

• MSR_paper_graphical_abstract
  [Text of the abstract for those who need it here: Text on MSR_paper_graphical_abstract]

For updated version of the graphical abstract see below.

NASA's Mars Sample Return team classified all except one of my attachments as "nonsubstantive" and so didn't look at them (NASA, 2023, MSR FINAL PEIS :B-28)

The only one they looked at was attachment 7 (Walker, 2022, So many serious mistakes in NASA's Mars Samples Environmental Impact Statement it needs a clean restart)

Their biggest omission was that they didn't look at attachment 8 (Walker, 2022, NASA and ESA are likely to be legally required to sterilize Mars samples to protect the environment until proven safe) which covered several topics in much greater depth than in the "so many serious mistakes" including quarantine, worst case scenarios and in situ instruments that could be used for studying the samples above GEO. Without looking at attachment 8 they had no way to evaluate the reasonable alternative of returning to a satellite above GEO properly.

These points are also covered in the first round of public comments - this is relevant because NASA had a legal obligation under NEPA to mention reasonable alternatives such as this in the draft EIS - I was puzzled that you never mentioned the major issues I raised of this reasonable alternative in the draft EIS - which is what led eventually to this open letter as a way to draw your attention to your legal obligation to take account of suggestions like this and issues raised by the public.

Are you aware of the ESF Mars Sample Return study (Ammann et al, 2012:14ff)? It said "The release of a single unsterilized particle larger than 0.05 μm is not acceptable under any circumstances”. This is to contain starvation limited ultramicrobacteria which pass through 0.1 micron filters (Miteva et al, 2005). Any Martian microbes may be starvation limited.

This 100% containment at 0.05 microns is well beyond capabilities of BSL4 facilities. Even ULPA level 17 filters only contain 99.999995 percent of particles tested only to 0.12 microns (BS, 2009:4).
...

The ESF also set a minimum one in a million probability of release of a single unsterilized particle at 0.01 microns. This is to contain gene transfer agents which transfer novel capabilities overnight in sea water to unrelated species of archaea (Maxmen, 2010)..

The ESF said both requirements need periodic review. This might reduce those figures further.

...

I propose two possible solutions in my article.

1. sterilize samples during the return journey, perhaps with nanoscale X-ray emitters. Present day life in the sample would be recognizable after sterilization OR

2. return unsterilized samples to a safe orbit where astrobiologists study them remotely using miniature instruments designed for life detection on Mars. Return sterilized sub-samples to Earth immediately;

As a safe orbit, this paper recommends the Laplace plane above GEO where ring particles would orbit if we had a ring system.

A return to the ISS doesn’t break the chain of contact with Mars.

The Moon needs to be kept free of contamination for future astronauts and tourists (COSPAR, 2011)

The preprint examines ways to increase chances of viable spores, such as dust samples (Jakovsky et al, 2021)

(Walker, 2022, Comment by posted on May 16th)

My comment on the first round of comments raises the issue of the ESF recommendation of 100% containment down to 0.05 microns, proposes sterilized return or return of samples to a miniature life detection lab above GEO and covers many of the other issues such as the issues with quarantine of technicians are covered in the attachment. See:

• (Walker, 2022, Comment posted on May 16th)
• (Preprint -NASA expected to be legally required to sterilize Mars samples to protect Earth's biosphere at least until 2039).
  [The title there refers to the time needed to complete a facility assuming a NEPA timeline of at least 7 years as I wasn't aware of the new streamliined one year NEPA process]

New version of the abstract I attached to the 20th December comment: (Walker, 2022, Comment posted December 20th)

Summary of rationale for suggested alternative:
- we can't use human technicians because of lab leaks
- because human quarantine can't keep out alien life
- a ground based telerobotic facility would likely cost over half a billion dollars
- we already have technology for in situ life detection as far away as Europa
- leading to suggested alternative: a telerobotic life detection lab above Geostationary Earth Orbit (GEO)

Video: Open letter to NASA - Reasonable alternative keeps Earth 100% safe - with greatly enhanced science

Reminder that you have a legal requirement to identify and do rigorous analysis of reasonable alternatives under NEPA § 1502.14. .

First here are the four bullet points in the title of this section with links:

1. we can't use human technicians because of lab leaks
   • Even the most well prepared biosafety laboratory has to have emergency procedures for lab leaks
      
2. because human quarantine can't keep out alien life
   • The Apollo missions used quarantine for technicians - but this was before NEPA and had no peer review - would not keep out mirror life or fungal diseases of crops or indeed humans - or diseases with life long symptomless human spreaders (similarly to typhoid Mary).
      
3. a ground based telerobotic facility would likely cost over half a billion dollars
   • ...unsterilized samples have to be returned to a telerobotic facility until we know what's there - but likely costs well over half a billion dollars...
      
4. we already have the capability to do in situ life detection as far away as Europa
   • NASA already have worked on plans for a remote life detection laboratory on Europa ...
      
5. leading to suggested alternative: a telerobotic life detection lab above Geostationary Earth Orbit (GEO)
   • We can eliminate all these issues and make it a far simpler mission using a miniature telerobotic facility above GEO

It's based on

Europa Lander life detection lab

NASA;s idea for Jupiter's moon Europa

Europa lander: total mass 42.5 kg

My suggestion: return samples to a miniature lab like this (adds centrifuge for artificial gravity)

But above GEO

Keeps Earth 100% safe from Mars microbes

Graphic from (NASA, 2017, Europa Lander Study 2016 Report)

Here is a longer summary in 25 points

Video: Open letter to NASA - Reasonable alternative keeps Earth 100% safe - with greatly enhanced science

1. NASA is not expert in public health or protecting the environment - and this is a topic where expertise in health is essential - they shouldn't have closed down the interagency panel or closed down their planetary protection office and need that expertise urgently - similarly for expertise in environmental effects, expertise in risk assurance, and expertise in communicating with the public - the EIS shows a lack of expertise in all these areas
    
2. This issue seems to be systemic, not the result of any particular administrator or scientist, but just because NASA is set up for engineering / space missions and not set up for planetary protection - and the organization itself has aligned with focusing more and more on the engineering and ignoring the planetary protection because it made the engineering more difficult and because it seemed to make their eventual aim to send humans to Mars more difficult
   • NASA has been world leading in planetary protection
     - but since early this century it moved the other direction against the advice of the Space Studies Board...
      
   • These are systemic errors due to having nobody on the team trained in planetary protection or risk assurance
     - not the fault of any individual scientist or engineer
     - Space Studies Board finds it always has to educate committee members unfamiliar with basic planetary protection concepts
      
      
3. There could be life in the samples - the iMost team even plan to test them for metabolism, respiration and see if they can get anything to grow
   • No we don't have evidence Mars is uninhabitable - you even plan to test Perseverance's samples to see if any lifeforms detected from Mars are still alive
      
4. The meteorite argument doesn't work - most terrestrial microbes couldn't survive the shock of ejection or the dehydration and cold of the vacuum of space.
   • Microbes from Jezero crater do NOT get to Earth better protected and faster in Mars meteorites
      
5. Those and other invalid arguments are the basis for NASA's false conclusion that they already know that environmental impacts would not be significant.
    
6. Likely low risk but of large-scale harm - as for a house fire - we do need to take care - need to look at worst case scenarios not just best case scenarios
   • experts do believe the risk of large-scale harm to human health or the environment is low
     - but even a low risk of this nature is HIGHLY SIGNIFICANT
      
7. Mars may be more habitable than most people think - the influential SR-SAG2 paints a picture of a much less habitable Mars than the more accurate Space Studies Board review of it - the EIS cites SR-SAG2 and doesn't cite the much less well known Space Studies Board review - this was commissioned because of a perception in some circles that the authors of SR-SAG2 were too closely aligned with NASA's Mars program office - a serious omission to leave it out of an EIS (probably just a mistake)
   • Space Studies Board review found many ways Jezero Crater could be more habitable than suggested in the review SR-SAG2 used in the EIS
      
8. The Space Studies Board review finds many knowledge gaps in SR-SAG2 and points to biofilms that can make desert microenvironments much more habitable, by setting up miniature "homes" for microbes, to potential for transport in the atmosphere, says there can be microenvironments we can't see from orbit, and says SR-SAG2 doesn't adequately discuss the implications of the seven types of potential microenvironments it said are possible on Mars.
   • that SR-SAG2 didn't adequately discuss transport in the atmosphere
      
   • that SR-SAG2 only briefly considered implications of lack of knowledge of microenvironments..
      
   • The Space Studies Review of SR-SAG2 draws special attention to biofilms ...
      
9. Vivid scenarios of independently evolved mirror life and a novel fungal genus like Aspergillus fumigatus - not adapted to humans to motivate space agencies to take their responsibilities seriously
   • Detailed illustrative mirror life scenario
      
   • Illustrative worst case scenario for human health and wildlife (especially birds): NOVEL GENUS OF FUNGI
      
10. We do need precautions for lab leaks - from WHO manual
    • Even the most well prepared biosafety laboratory has to have emergency procedures for lab leaks
       
11. We can't use quarantine of technicians to keep out mirror life or novel fungi
    • The Apollo missions used quarantine for technicians - but this was before NEPA and had no peer review - would not keep out mirror life or fungal diseases of crops or indeed humans - or diseases with life long symptomless human spreaders (similarly to typhoid Mary)
       
12. So we have to return to a telerobotic facility (telerobots can be sterilized unlike human technicians)
     
13. By the ESF study we also have to keep out ultramicrobacteria and gene transfer agents, well beyond the capabilities of a BSL-4
    • The European Space Foundation in 2012 said we have to contain the far smaller ultramicrobacteria and gene transfer agents - ... well beyond capabilities of a BSL-4
       
14. A ground based telerobotic facility would cost a lot (well over half a billion dollars for sure) and be difficult technically and take a long time to be ready based on the designs NASA commissioned last time it was looking for Mars Sample Return Facilities - and that was for BSL-4 level of containment
    • ...unsterilized samples have to be returned to a telerobotic facility until we know what's there - but likely costs well over half a billion dollars...
       
15. We already know how to do life detection as far away as Europa
    • NASA already have worked on plans for a remote life detection laboratory on Europa ...
       
16. leading to suggested alternative: a telerobotic life detection lab in a safe orbit above Geostationary Earth Orbit (GEO)
    • We can eliminate all these issues and make it a far simpler mission using a miniature telerobotic life detection lab above GEO
       
17. This keeps Earth 100% safe
     
18. We can't do safety testing - we don't have this dichotomy that it's either no life or lots of life - Mars might have very low numbers of microbes by the Space Studies Board review of SR-SAG2 and potential for small patches of life in microenvironments or small amounts of life spread in the dust
    • Safety testing in an orbital facility can't be used at all if microbes occur in very low concentrations of only a few microbes per sample...
       
19. Humans go nowhere near and we sterilize anything returned to Earth
    • We should be able to sterilize even unknown Martian exobiology by using levels of ionizing radiation that will break the covalent bonds that hold complex chemicals together
      - 100 million years equivalent of surface ionizing radiation may be sufficient but this needs expert attention in a multi-disciplinary approach involving experts in synthetic life and possibilities for alternative evolutionary pathways
      - no effect on isotope ratios and virtually no changes for geology
          
       
20. We need bonus samples in clean containers sent with the ESA fetch rover because the Perseverance samples aren't clean enough for astrobiology (another effect of NASA's engineering focus)
    • But we can rescue this - with bonus samples in CLEAN containers sent on the EIS for astrobiology - a thimbleful of dirt, a few grams of dust, clean atmospheric samples, and a representative pebble from a recently excavated crater
       
21. We must be careful not to raise expectations too high. This is just the second step in Carl Sagan's "vigorous program" after Viking. Results may be just as ambiguous for this second step as for the first step
    • Even with bonus samples, it is important to present this to the public as just the second step in Carl Sagan's "Vigorous program of unmanned exobiology"...
       
22. But we can make rapid progress if we pivot to make 100% sterile landers and rovers and send 100s of them in one mission in near future missions to search for life in situ throughout Mars with new technology that can easily be sterilized with a few minutes at 300°C or higher,
    • The best way to achieve Sagan's "vigorous program of unmanned exobiology" is to use 100% sterile landers and rovers - and we have the technology now to do it with HOTTech
       
23. This will help space colonization enthusiasts too - they need hard science rather than vivid metaphors to convince the rest of us that there is nothing by way of mirror life or alien fungi on Mars or anything potentially harmful
    • Understandably those focused on landing human astronauts on the Martian surface think this can happen sooner if we drop all planetary protection
      - but they need to convince the rest of us their intuitions of a biologically harmless Mars are correct
       
    • With clean missions to Mars and clean samples returned from Mars, we get a clearer picture of what’s on Mars far faster
       
24. If we do have a mirror life terrestrial planet in our solar system - this would hugely increase interest in space exploration and be a stimulus for humans in space and for humans orbiting Mars, living on its moons, and exploring other places throughout the solar system - even though it would likely mean humans never live on the surface.
    • The Moon is hard to get to and far more interesting than we realized in the 1960s and 1970s - many adventures there before we go further - Chris Hadfield - former commander of the ISS thinks ultimately we will be living on the Moon for a generation before we go to Mars - "It’s as if you and I were in Paris, paddling around in the Seine in little canoes saying, 'We’ve got boats, we’ve got paddles, let’s go to Australia!' Australia? We can barely cross the English Channel."
       
    • If we have a mirror life terrestrial planet in our solar system - this will surely lead to more not less interest in space exploration and settlement - we'd need to travel light years to find another one
       
    • The spectacular HERRO orbit for our first humans in Mars orbit controlling assets on the ground including telerobotic avatars
       
    • Next step as our spacecraft get more capable - Jupiter's moon Callisto - far better for humans than Europa, naturally shielded from ionizing radiation and no planetary protection issues either way as it is classified similarly to the Moon for planetary protection
       
    • Then Titan - the only Moon with an atmosphere, 1.5 times Earth's atmospheric pressure - - far easier to protect against cold than vacuum - probably no planetary protection issues either way if it doesn't have cryovolcanism - so exceptionally cold any life there is unlikely to be able to survive on Earth
       
    • Finally with space habitats slowly spinning for artificial gravity and large thin film mirrors we can colonize out to Pluto and beyond
       
25. We can likely even grow plants on the surface of a mirror life planet - and with no risk of forwards contamination either - as seeds can be sterilized
    • ... if they have to stay in orbit - they can use those assets to exploit Mars and even set up robotic farms on the surface which would work even on a mirror life planet because seeds can be sterilized unlike humans

The satellite itself with the centrifuge:

Text on graphic: Bonus samples in STERILE containers returned to satellite perhaps 50,000 or 100,000 km above GEO in what would be Earth’s ring plane if it had a ring system.

• NOT for safety testing
• Returned for astrobiological study – nexus of expanding off-planet astrobiology lab.
• Minimal forward contamination.
• Humans nowhere near this.
• Centrifuge to replicate martian gravity.

Many instruments placed in centrifuge along with the dust and operated remotely from Earth. Illustration shows:

• Chiral labeled release.
• SETG from sample acquisition through to DNA sequence all automated in 2 units, each can be held in palm of hand.
• Astrobionibbler microfluidics can detect a single amino acid in a gram of sample

This would be minimal cost for NASA as the instruments would be funded by universities.

Graphic shows: (NOAA’s new GOES-17 weather satellite has degraded vision at night) just to have an image of a geostationary satellite, not that it would be a $2.5 billion dollar satellite. SETG from (Mojarro et al., 2016, SETG: nucleic acid extraction and sequencing for in situ life detection on Mars). Astrobionibbler from (Elleman, 2014, Path to Discovery) ISS centrifugal motor for plant experiments, dialable to any level from microgravity to 2g (Centrifuge Rotor [biology experiment on the ISS])