Statement

NASA-STD-8719.27 §5.4.2.b requires sterilization and inactivation of bioactive molecules prior to Earth-Moon System entry, leveraging overkill levels of terrestrial sterilization. The standard assumes extraterrestrial biological systems would be susceptible to the same inactivation mechanisms as terrestrial systems. Prion-like entities (PLEs) represent the hardest case: no nucleic acid target, conformationally stable protein aggregates resistant to conventional sterilization. Gominet et al. (2007) hypothesized a 4-5 log reduction (based on infection rates and incubation time) on fresh, protected terrestrial prions at 50 kGy Co-60 gamma in an inert matrix. Mars samples present worse conditions for PLE survival: PLEs would be pre-degraded by exposure to cosmic radiation and perchlorate radiolysis products, and hosted in a mineral matrix which may amplify radiolytic damage 21-35× (Pavlov et al., 2022). We claim that under Jezero/MSR conditions 50 kGy is sufficient to inactivate prions in native materials and that the 100 kGy reference dose provides overkill margin consistent with §5.4.2.b.

Inactivation definition: A prion-like entity is inactivated when it loses the conformational integrity required to catalyze pathogenic protein misfolding in a susceptible host.

Open experimental questions: whether mineral matrix radiolytic enhancement applies to prion inactivation at the same magnitude as small-molecule radiolysis, and the effectiveness of perchlorate-derived oxidants against desiccated protein aggregates, whether via transient deliquescence events (for solution-phase hypochlorite) or chronic gas-phase exposure (for chlorine dioxide). See Implications section.

Evidence

E1: Gominet et al. 2007, Radiation Physics and Chemistry 76(11-12):1760-1762. DOI:10.1016/j.radphyschem.2007.02.099
Co-60 gamma irradiation of scrapie prions (strain C506M3) at ~1 kGy/h.

• Matrix: brain homogenate in fetal bovine serum, dried on glass tubes.
• Bioassay: intracerebral inoculation of C57Bl/6 mice, 24 mice per dose level.
• Starting titer: >10^6.3 LD50.
• Results from undiluted control baseline (100% transmission, 170-day incubation):
  • 25 kGy: 83% transmission (20/24), 229-day incubation.
  • 50 kGy: 58% transmission (14/24), 235-day incubation.
  • 100 kGy: 67% transmission (16/24), 290-day incubation.
• Transmission reduction is partial and non-monotonic across dose. Incubation period is the only endpoint that increases consistently with dose. Authors hypothesized 4-5 log reduction at 50 kGy based on comparison with dilution series; however, the 10^-5 dilution control showed 75% transmission at 232-day incubation, which is not cleanly distinguishable from the 50 kGy result.
• Author conclusion: gamma "cannot be considered as a sterilizing agent able to ensure a total prion inactivation."

E2: Terrestrial prion persistence limits
Brown & Gajdusek 1991 (Lancet 337:269-270, DOI:10.1016/0140-6736(91)90873-N): Scrapie survived ≥3 years burial in temperate garden soil with 2-3 log reduction from ~5 log input titer. Somerville et al. 2019 (Arch Virol 164:1135-1145, DOI:10.1007/s00705-019-04154-8): Mouse-passaged BSE survived ≥5 years burial in clay and sandy soil with no decline trend across annual exhumations. Georgsson et al. 2006 (J Gen Virol 87:3737-3740, DOI:10.1099/vir.0.82011-0): Scrapie persisted ≥16 years in an undisinfected sheep-house, producing 58% subclinical infection in a newly exposed flock. All persistence data from biologically active environments with moisture, microbial soil flora, and protective organic matrices.

E3: Prion-mineral binding
Johnson et al. 2006 (PLoS Pathog 2(4):e32, DOI:10.1371/journal.ppat.0020032): PrPTSE binds rapidly and strongly to montmorillonite, kaolinite, and quartz. Mineral-bound prions retain full infectivity.
Johnson et al. 2007 (PLoS Pathog 3:e93, DOI:10.1371/journal.ppat.0030093): Montmorillonite binding increased oral disease penetrance 680× relative to unbound agent. Under Earth conditions, mineral binding protects prion structure.

E4: Mars surface degradation mechanisms
Mars regolith functions as a continuous oxidant-generating system. Perchlorate has been detected at concentrations of 0.05-1.05 wt% at every Gale Crater sampling site (Sutter et al. 2017, JGR: Planets 122:2574-2609, DOI:10.1002/2016JE005225), consistent with the 0.4-0.6 wt% measured by Phoenix at Vastitas Borealis (Hecht et al. 2009, Science 325:64-67, DOI:10.1126/science.1172466). Under galactic cosmic ray bombardment, perchlorate radiolysis produces hypochlorite (G = 0.032 ± 0.005 molecules/100 eV), chlorine dioxide (G = 0.109 ± 0.007), and molecular oxygen (G = 1.6 ± 0.2) (Quinn et al. 2013, Astrobiology 13:515-520, DOI:10.1089/ast.2013.0999). Perchlorate is continuously reformed via an atmospheric pathway involving OClO sublimation from ices and gas-phase oxidation (Wilson et al. 2016, JGR: Planets 121:1472-1491, DOI:10.1002/2016JE005078), establishing a steady-state oxidant reservoir rather than a depleting one.

This oxidant-generating system has operated over geological timescales. The measured surface GCR dose rate is 76 ± 14 mGy/yr (Hassler et al. 2014, Science 343:1244797, DOI:10.1126/science.1244797). The youngest surface exposure age measured on Mars is 78 ± 30 Myr for Cumberland mudstone at Gale Crater (Farley et al. 2014, Science 343:1247166, DOI:10.1126/science.1247166); Jezero crater floor rocks have crater-counting ages of 1.4-2.5 Ga (Zorzano et al. 2026, Scientific Reports 16:3884, DOI:10.1038/s41598-025-33031-5). At 0.5 wt% perchlorate and the measured GCR flux, the accumulated radiolysis energy over 78 Myr is sufficient to destroy the entire perchlorate inventory approximately 7 times; over 1 Gyr, approximately 100 times. The perchlorate observed today is not ancient inventory but the instantaneous snapshot of a dynamic system where radiolytic destruction is balanced by atmospheric reformation. Each turnover cycle generates reactive chlorine species. When gamma radiation strikes silicate minerals, the minerals themselves generate radicals and secondary electrons; these reactive species then migrate to and attack organic molecules adsorbed on mineral surfaces. Pavlov et al. (2022, Astrobiology 22:1099-1115, DOI:10.1089/ast.2021.0166) demonstrated this effect quantitatively: amino acids in fused silica matrices are destroyed 21× faster than pure amino acids under gamma irradiation, and with sodium perchlorate present, 35× faster. The damaging radiolysis occurs in the mineral matrix, not in the organic molecule itself.

Jezero ground temperatures reach 290 K (17°C) during daytime, with diurnal swings exceeding 100 K (Rodriguez-Manfredi et al. 2023, Nature Geoscience 16:19-28, DOI:10.1038/s41561-022-01084-0). Calcium perchlorate deliquesces at ≤50% Relative Humidity (RH) across all temperatures studied (223–273 K), with DRH as low as ~26% for the tetrahydrate at 253 K and ~1% for anhydrous Ca(ClO₄)₂ at 273 K; once deliquesced, solutions persist as metastable supersaturated brines until RH drops to ~15% on average (Nuding et al. 2013, 44th LPSC Abstract #2584; Nuding et al. 2014, Icarus 243:420-428, DOI:10.1016/j.icarus.2014.08.044). Jezero nighttime RH reaches ~29% (Rodriguez-Manfredi et al. 2023), which exceeds the hydrated-salt DRH at evening temperatures when the surface is still well above the Ca(ClO₄)₂ eutectic (~198 K). This leads to transient thin brine films forming on grain surfaces during diurnal humidity cycles, with the hysteresis effect extending brine persistence into drier daytime conditions.

Of the perchlorate radiolysis products, chlorine dioxide (ClO2) warrants separate treatment. Unlike hypochlorite, which requires a solvent medium to contact proteins, ClO2 is a gaseous free radical with one unpaired electron. It oxidizes organic compounds without spin barriers, unlike triplet O2 which is kinetically unreactive at Mars temperatures (Gobi et al. 2016, Astrophys. J. 832:164, DOI:10.3847/0004-637X/832/2/164). ClO2 is generated continuously on the Mars surface by two independent pathways: UV photolysis of perchlorate/chlorate (Newmark & Kounaves 2024, Sci. Rep. 14:7682, DOI:10.1038/s41598-024-57968-1) and GCR radiolysis of perchlorate (Quinn et al. 2013, G = 0.109 ± 0.007 molecules/100 eV, approximately 3.4× higher than hypochlorite production). Being gaseous, ClO2 permeates through regolith and contacts organic compounds below the UV-exposed surface without requiring liquid water. Newmark & Kounaves (2024) demonstrated >95% destruction of alanine coated on silica sand in 24 h at 22°C/25% RH, and approximately 46% destruction at -15°C/8.5% RH, with the ClO2 separated from the alanine by a glass microfiber barrier. Dineen et al. (2026, 57th LPSC, Abstract #1305) extended this to complex biomolecules: chlorophyll-b and nickel tetraphenylporphine showed degradation products within 20 minutes at sub-1 ppm ClO2 concentration under Mars simulation conditions. ClO2 denatures proteins by covalent oxidative modification of tryptophan and tyrosine residues, transferring oxygen atoms from ClO2 itself rather than from water: tryptophan is converted to N-formylkynurenine (+32 Da) and tyrosine to DOPA or TOPA (+16 Da), both irreversibly (Ogata 2007, Biochemistry 46:4898-4911, DOI:10.1021/bi061827u). Oxidation of a single surface-exposed tryptophan residue (W153) in influenza haemagglutinin abolishes receptor-binding function, with IC50 = 13.7 uM at 25°C for 2 min and half-life of 19.5 s at 100 uM (Ogata 2012, J. Gen. Virol. 93:2558-2563, DOI:10.1099/vir.0.044263-0). Accessibility governs which residues are attacked; buried residues in tightly packed protein structures are shielded from ClO2 while surface-exposed residues are preferentially oxidized (Ogata 2007, 2012).

E5: Solid-state protein radiolysis mechanisms
Audette-Stuart et al. 2005 (Radiat. Phys. Chem. 72:301-306, DOI:10.1016/j.radphyschem.2003.12.060): Gamma irradiation of beta-galactosidase in liquid, frozen, and lyophilized states with selective radical scavengers. In the frozen and lyophilized states, an OH radical scavenger (Tris, 1000:1 ratio) provided no protection against protein fragmentation or enzyme inactivation. An electron scavenger (NaNO3, 1000:1 ratio) reduced fragmentation yield by approximately 33%. Fragmentation yield in the solid state was approximately 24× lower than in dilute liquid solution. The quantity of water present did not affect solid-state fragmentation yields. Authors conclude that OH radicals do not contribute significantly to protein damage in the solid state; electrons from water molecules closely associated with the protein surface are the primary species causing peptide chain cleavage in frozen and lyophilized systems.
Kempner 2011 (J. Polym. Sci. B Polym. Phys. 49:827-831, DOI:10.1002/polb.22250): In dry or frozen states, virtually all radiation damage to macromolecules results from direct ionization. Each ionization event irreversibly breaks covalent bonds. Larger molecules are more likely to suffer ionization events per unit dose.
Garrison 1987 (Chem. Rev. 87:381-398, DOI:10.1021/cr00078a006): Comprehensive review of peptide and protein radiolysis in both aqueous and solid-state systems. Solid-state radiolysis produces backbone cleavage via alpha-carbon radical formation, reductive deamination, and side-chain modification with established G-values for dry amino acids and polypeptides.

E6: Jezero sample mineralogy
Jezero delta sediments contain Fe/Mg-smectite clays (nontronite, saponite), hydrated sulfates, carbonates, serpentine, and amorphous phases (Farley et al. 2022, Science 377:abo2196, DOI:10.1126/science.abo2196; Bosak et al. 2024, AGU Adv. 5:e2024AV001241). Analytical totals for delta outcrops range from 83.5 to 94.5 wt%, indicating 5.5-16.5 wt% unmeasured volatiles (H2O, CO2, organics). Smectite interlayer water is held by cation hydration energy and requires heating to 300-800 C for release; it persists under Mars ambient conditions and within sealed sample tubes. Zorzano et al. (2025, Astrobiology 25(10):725-741, DOI:10.1177/15311074251382585) estimate 5-20× radiolysis enhancement for these lithologies based on mineral matrix composition, and note: "The same properties that preserve organics (fine-grained, hydrated minerals, high surface area) also enhance radiolysis."

Argument

A1: Gominet demonstrates partial inactivation of fresh, protected prions by gamma alone.
Even under worst-case conditions (fresh brain homogenate, inert matrix, no environmental degradation), Co-60 gamma reduced transmission from 100% to 58-67% and extended incubation by 35-71%. Authors hypothesized 4-5 log reduction; the data support significant but incomplete inactivation. This is the baseline that the Mars environmental arguments improve upon.

A2: Mars presents chemical attack mechanisms absent from terrestrial soil.
Terrestrial prion persistence (E2) was measured in biologically active environments with moisture, microbial soil flora, and protective organic matrices. Mars has none of these.

A3: Perchlorate radiolysis products contribute to PLE degradation during surface residence.
Mars regolith contains 0.4-0.6% perchlorate by mass (Hecht et al. 2009). Gamma irradiation of perchlorate produces hypochlorite that chlorinates and decomposes amino acids (Quinn et al. 2013). However, hypochlorite-protein reactions are solution-phase chemistry requiring a solvent medium. In the desiccated Jezero regolith, the pathway for oxidant-protein contact is perchlorate deliquescence: transient thin brine films forming on grain surfaces during diurnal humidity cycles (E4). Nuding et al. (2014) measured the DRH of hydrated Ca(ClO₄)₂ at ~26% at 253 K; Rodriguez-Manfredi et al. (2023) measured Jezero nighttime RH reaching ~29%. The humidity exceeds the DRH at relevant evening temperatures, and the large hysteresis (ERH ~15%) means brine films persist well into the drying phase of each cycle. Over a notional 14-18 year surface residence period (M2020 landing through projected sample return Earth-entry), the cumulative number of deliquescence cycles is non-trivial. The chemical effectiveness of transient brine contact against a desiccated protein aggregate is unquantified, but the conditions for brine formation are met and the exposure time is long. Perchlorate radiolysis products have accumulated in the regolith over geological time; a PLE deposited during the Mars 2020 landing would have contacted this existing oxidant reservoir at the next deliquescence event, not after a new accumulation period. Terrestrial prions persist at least 16 years in soil (Georgsson et al. 2006), so time alone does not inactivate PLEs within this window, but intermittent oxidant exposure during deliquescence events contributes partial degradation prior to the sterilization phase during sample return.

A second perchlorate radiolysis product, chlorine dioxide (ClO2), does not require solvent access. ClO2 is a gas-phase radical generated continuously by UV photolysis and GCR radiolysis of perchlorate (E4). It permeates through regolith as a vapor and contacts adsorbed organics directly, without needing a deliquescence event. Over the 14-18 year surface residence period (if prions were released during the M2020 landing), continuous low-level ClO2 exposure would progressively oxidize surface-accessible tryptophan and tyrosine residues on a PLE aggregate. Each oxidation event is irreversible: tryptophan becomes N-formylkynurenine, tyrosine becomes DOPA or TOPA (Ogata 2007). In influenza virus, oxidation of a single surface-exposed tryptophan residue abolishes protein function (Ogata 2012). Prion fibrils present a tightly packed beta-sheet core that shields interior residues. ClO2 attack would proceed outside-in, progressively oxidizing residues at the seeding interface while the core remains temporarily protected. No gas-phase kinetics exist for ClO2 against desiccated protein aggregates at any concentration or timescale. Mars surface ClO2 concentration is unquantified; Dineen et al. (2026) measured sub-1 ppm in a laboratory Mars simulation. The chronic low-concentration gas-phase regime over 14-18 years is completely unstudied. The mechanism is real, the chemistry is irreversible, and the exposure time is long, but the rate of PLE inactivation under these conditions has not been quantified.

A4: Mineral matrix radiolysis enhances sterilization during the 22-month return trip.
Mars samples are mineral-hosted by definition. Silicate matrices amplify radiolytic damage 21× over pure compounds; with perchlorate present, 35× (Pavlov et al. 2022). The mechanism involves secondary electrons and radicals generated in the mineral matrix migrating to adsorbed organic molecules. Audette-Stuart et al. (2005) demonstrated that electrons from water molecules at the protein-solvent interface contribute approximately 33% of protein fragmentation yield in the solid state, and that OH radicals do not contribute to solid-state protein damage (E5). This establishes that electron-mediated damage from bound water operates on proteins, not just on the small molecules Pavlov tested. Jezero delta samples contain Fe/Mg-smectite clays with structural interlayer water that persists under Mars conditions and within sealed sample tubes (E6). During Cs-137 sterilization, this mineral-hosted water provides electrons for protein fragmentation via the same mechanism Audette-Stuart demonstrated, amplified by the Pavlov mineral matrix effect. Gominet's prions were dried on glass tubes (an inert, non-radical-generating matrix). Jezero Fe-smectite is the opposite: an active radical source under irradiation with 5-20× estimated enhancement (Zorzano et al. 2025). Under ambient conditions, mineral binding protects prion structure and enhances infectivity (Johnson et al. 2006, 2007). We are arguing that under active gamma irradiation, the same mineral surfaces that confer protection become sources of radical production which attack prion protein structures.

See also Mineral Matrix Enhancement of Organic Radiolysis.

Implication

Gominet's hypothesized 4-5 log reduction at 50 kGy is not a Mars-analog study. Their prions were fresh, protected, and irradiated in an inert matrix. Mars samples present the opposite conditions: pre-degraded material in a radiolysis-enhancing mineral matrix. We argue that under Jezero/MSR conditions 50 kGy is sufficient to inactivate prions in Mars materials and that the 100 kGy reference dose provides overkill margin consistent with NASA-STD-8719.27 §5.4.2.b.

Re: Open experimental questions. Three remain. First, whether mineral matrix radiolytic enhancement applies to protein aggregates at the same 21-35× magnitude Pavlov measured for amino acids. Audette-Stuart et al. (2005) established that electron-mediated damage from bound water operates on proteins in the solid state, so the mechanism is not hypothetical, but the scaling factor is. Second, the effectiveness of perchlorate-derived oxidants against desiccated protein aggregates during transient deliquescence events has not been studied. Third, gas-phase chlorine dioxide degradation kinetics against desiccated protein aggregates at Mars-relevant concentrations and timescales have not been measured. All three are tractable experiments. We're going to focus on constrained D. radiodurans under low-dose-rate gamma for one year instead (a much more logical/concerning model organism). See 100 kGy Gamma Sterilization Inactivates D. radiodurans. We don't currently plan to put D. radiodurans in a geologic or environmental context either, so that's another good one. Forward this to your post-docs!

The author notes that this claim is not in the report yet, but impacts on biosignature detection are expected and will percolate through eventually.

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