Chemical compounds causing severe acute toxicity in heavy liquids used for intraocular surgery.

Perfluorocarbon liquids (PFCLs) have been considered safe for intraocular manipulation of the retina, but since 2013 many cases of acute eye toxicity cousing blindness have been reported in various countries when using various commercial PFCLs. All these PFCLs were CE marked (Conformité Européenne), which meant they had been subjected to evaluation complying with the International Organization for Standardization (ISO) guidelines. These dramatic events raised questions about the safety of PFCLs and the validity of some cytotoxicity tests performed under ISO guidelines. Samples from toxic batches were analyzed by gas chromatography-mass spectrometry combined with Raman and infrared spectrometry. Perfluorooctanoic acid, dodecafluoro-1-heptanol, ethylbenzene and tributyltin bromide were identified and evaluated by a direct contact cytotoxicity test using ARPE-19 cell line, patented by our group (EP 3467118 A1). Perfluorooctanoic acid at a concentration of >0.06 mM and tributyltin bromide at a concentration of ≥0.016 mM were shown to be toxic, whereas the concentration found in the toxic samples reached 0.48 mM, and 0.111 mM, respectively. These finding emphasized the idea that determination of partially fluorinated compounds are not enough to guarantee the safety of these medical devices.

ACUTE RETINAL DAMAGE AFTER USING A TOXIC PERFLUORO-OCTANE FOR VITREO-RETINAL SURGERY.

PURPOSE: To describe a series of retinal acute toxicity cases with severe visual loss after intraocular use of a toxic perfluoro-octane (PFO). The clinical presentation is described, and the likely causes are analyzed. New biological methods for testing safety of intraocular medical devices are proposed. METHODS: Information regarding a series of eyes suffering acute severe events after intraocular use of a toxic PFO was analyzed. Four types of spectroscopy, nuclear magnetic resonance, and chromatography were used to identify the potential PFO contaminants. Cultures of human retinal pigment epithelial cells (ARPE-19) and porcine neuroretina were used to quantify the toxicity of the suspect PFO lots. RESULTS: Of 117 cases of intraocular toxicity, 96 were considered clearly related to the use of PFO. Fifty-three cases had no light perception, and 97 had no measurable visual acuity. Retinal necrosis (n = 38) and vascular occlusion (n = 33) were the most characteristic findings. Two hydroxyl compounds, perfluorooctanoic acid and dodecafluoro-1-heptanol, and benzene derivatives were identified as the suspected toxic agents. While existing toxicity testing failed, we proposed new tests that demonstrated clear toxicity. CONCLUSION: Protocols to determine cytotoxicity of intraocular medical devices should be revised to assure safety. Acute toxic events should be reported to health authorities and scientific media.

Dark microbiome and extremely low organics in Atacama fossil delta unveil Mars life detection limits.

Identifying unequivocal signs of life on Mars is one of the most important objectives for sending missions to the red planet. Here we report Red Stone, a 163-100 My alluvial fan-fan delta that formed under arid conditions in the Atacama Desert, rich in hematite and mudstones containing clays such as vermiculite and smectites, and therefore geologically analogous to Mars. We show that Red Stone samples display an important number of microorganisms with an unusual high rate of phylogenetic indeterminacy, what we refer to as "dark microbiome", and a mix of biosignatures from extant and ancient microorganisms that can be barely detected with state-of-the-art laboratory equipment. Our analyses by testbed instruments that are on or will be sent to Mars unveil that although the mineralogy of Red Stone matches that detected by ground-based instruments on the red planet, similarly low levels of organics will be hard, if not impossible to detect in Martian rocks depending on the instrument and technique used. Our results stress the importance in returning samples to Earth for conclusively addressing whether life ever existed on Mars.

Spectroscopic study of terrestrial analogues to support rover missions to Mars - A Raman-centred review.

The 2020s could be called, with little doubt, the "Mars decade". No other period in space exploration history has experienced such interest in placing orbiters, rovers and landers on the Red Planet. In 2021 alone, the Emirates' first Mars Mission (the Hope orbiter), the Chinese Tianwen-1 mission (orbiter, lander and rover), and NASA's Mars 2020 Perseverance rover reached Mars. The ExoMars mission Rosalind Franklin rover is scheduled for launch in 2022. Beyond that, several other missions are proposed or under development. Among these, MMX to Phobos and the very important Mars Sample Return can be cited. One of the key mission objectives of the Mars 2020 and ExoMars 2022 missions is the detection of traces of potential past or present life. This detection relies to a great extent on the analytical results provided by complementary spectroscopic techniques. The development of these novel instruments has been carried out in step with the analytical study of terrestrial analogue sites and materials, which serve to test the scientific capabilities of spectroscopic prototypes while providing crucial information to better understand the geological processes that could have occurred on Mars. Being directly involved in the development of three of the first Raman spectrometers to be validated for space exploration missions (Mars 2020/SuperCam, ExoMars/RLS and RAX/MMX), the present review summarizes some of the most relevant spectroscopy-based analyses of terrestrial analogues carried out over the past two decades. Therefore, the present work describes the analytical results gathered from the study of some of the most distinctive terrestrial analogues of Martian geological contexts, as well as the lessons learned mainly from ExoMars mission simulations conducted at representative analogue sites. Learning from the experience gained in the described studies, a general overview of the scientific outcome expected from the spectroscopic system developed for current and forthcoming planetary missions is provided.

Planetary Terrestrial Analogues Library Project: 3. Characterization of Samples With MicrOmega.

The Planetary Terrestrial Analogues Library (PTAL) project aims at building and exploiting a database involving several analytical techniques, to help characterize the mineralogical evolution of terrestrial bodies, starting with Mars. Around 100 natural Earth rock samples have been collected from selected locations to gather a variety of analogs for martian geology, from volcanic to sedimentary origin with different levels of alteration. All samples are to be characterized within the PTAL project with different mineralogical and elemental analysis techniques, including techniques brought on actual and future instruments at the surface of Mars (near infrared [NIR] spectroscopy, Raman spectroscopy, and laser-induced breakdown spectroscopy). This article presents the NIR measurements and interpretations acquired with the ExoMars MicrOmega spare instrument. MicrOmega is an NIR hyperspectral microscope, mounted in the analytical laboratory of the ExoMars rover Rosalind Franklin. All PTAL samples have been observed at least once with MicrOmega using a dedicated setup. For all PTAL samples, data description and interpretation are presented. For some chosen examples, color composite images and spectra are presented as well. A comparison with characterizations by NIR and Raman spectrometry is discussed for some of the samples. In particular, the spectral imaging capacity of MicrOmega allows detections of mineral components and potential organic molecules that were not possible with other one-spot techniques. In addition, it enables estimation of heterogeneities in the spatial distribution of various mineral species. The MicrOmega/PTAL data shall support the future observations and analyses performed by MicrOmega/Rosalind Franklin instrument.

Organic geochemistry and mineralogy suggest anthropogenic impact in speleothem chemistry from volcanic show caves of the Galapagos.

The network of lava tubes is one of the most unexploited natural wonders of the Galapagos Islands. Here, we provide the first morphological, mineralogical, and biogeochemical assessment of speleothems from volcanic caves of the Galapagos to understand their structure, composition, and origin, as well as to identify organic molecules preserved in speleothems. Mineralogical analyses revealed that moonmilk and coralloid speleothems from Bellavista and Royal Palm Caves were composed of calcite, opal-A, and minor amounts of clay minerals. Extracellular polymeric substances, fossilized bacteria, silica microspheres, and cell imprints on siliceous minerals evidenced microbe-mineral interactions and biologically-mediated silica precipitation. Alternating depositional layers between siliceous and carbonate minerals and the detection of biomarkers of surface vegetation and anthropogenic stressors indicated environmental and anthropogenic changes (agriculture, human waste, and cave visits) on these unique underground resources. Stable isotope analysis and Py-GC/MS were key to robustly identify biomarkers, allowing for implementation of future protection policies.

Raman Characterization of the CanMars Rover Field Campaign Samples Using the Raman Laser Spectrometer ExoMars Simulator: Implications for Mars and Planetary Exploration.

The Mars 2020 Perseverance rover landed on February 18, 2021, and has started ground operations. The ExoMars Rosalind Franklin rover will touch down on June 10, 2023. Perseverance will be the first-ever Mars sample caching mission-a first step in sample return to Earth. SuperCam and Scanning Habitable Environments with Raman & Luminescence for Organics & Chemicals (SHERLOC) on Perseverance, and Raman Laser Spectrometer (RLS) on Rosalind Franklin, will comprise the first ever in situ planetary mission Raman spectroscopy instruments to identify rocks, minerals, and potential organic biosignatures on Mars' surface. There are many challenges associated when using Raman instruments and the optimization and quantitative analysis of resulting data. To understand how best to overcome them, we performed a comprehensive Raman analysis campaign on CanMars, a Mars sample caching rover analog mission undertaken in Hanksville, Utah, USA, in 2016. The Hanksville region presents many similarities to Oxia Planum's past habitable conditions, including liquid water, flocculent, and elemental compounds (such as clays), catalysts, substrates, and energy/food sources for life. We sampled and conducted a complete band analysis of Raman spectra as mission validation analysis with the RLS ExoMars Simulator or RLS Sim, a breadboard setup representative of the ExoMars RLS instrument. RLS Sim emulates the operational behavior of RLS on the Rosalind Franklin rover. Given the high fidelity of the Mars analog site and the RLS Sim, the results presented here may provide important information useful for guiding in situ analysis and sample triage for caching relevant for the Perseverance and Rosalind Franklin missions. By using the RLS Sim on CanMars samples, our measurements detected oxides, sulfates, nitrates, carbonates, feldspars, and carotenoids, many with a higher degree of sensitivity than past results. Future work with the RLS Sim will aim to continue developing and improving the capability of the RLS system in the future ExoMars mission.

ExoFiT trial at the Atacama Desert (Chile): Raman detection of biomarkers by representative prototypes of the ExoMars/Raman Laser Spectrometer.

In this work, the analytical research performed by the Raman Laser Spectrometer (RLS) team during the ExoFiT trial is presented. During this test, an emulator of the Rosalind Franklin rover was remotely operated at the Atacama Desert in a Mars-like sequence of scientific operations that ended with the collection and the analysis of two drilled cores. The in-situ Raman characterization of the samples was performed through a portable technology demonstrator of RLS (RAD1 system). The results were later complemented in the laboratory using a bench top RLS operation simulator and a X-Ray diffractometer (XRD). By simulating the operational and analytical constraints of the ExoMars mission, the two RLS representative instruments effectively disclosed the mineralogical composition of the drilled cores (k-feldspar, plagioclase, quartz, muscovite and rutile as main components), reaching the detection of minor phases (e.g., additional phyllosilicate and calcite) whose concentration was below the detection limit of XRD. Furthermore, Raman systems detected many organic functional groups (-C≡N, -NH2 and C-(NO2)), suggesting the presence of nitrogen-fixing microorganisms in the samples. The Raman detection of organic material in the subsurface of a Martian analogue site presenting representative environmental conditions (high UV radiation, extreme aridity), supports the idea that the RLS could play a key role in the fulfilment of the ExoMars main mission objective: to search for signs of life on Mars.

ExoMars Raman Laser Spectrometer: A Tool to Semiquantify the Serpentinization Degree of Olivine-Rich Rocks on Mars.

We evaluated the effectiveness of the ExoMars Raman laser spectrometer (RLS) to determine the degree of serpentinization of olivine-rich units on Mars. We selected terrestrial analogs of martian ultramafic rocks from the Leka Ophiolite Complex (LOC) and analyzed them with both laboratory and flight-like analytical instruments. We first studied the mineralogical composition of the samples (mostly olivine and serpentine) with state-of-the-art diffractometric (X-ray diffractometry [XRD]) and spectroscopic (Raman, near-infrared spectroscopy [NIR]) laboratory systems. We compared these results with those obtained using our RLS ExoMars Simulator. Our work shows that the RLS ExoMars Simulator successfully identified all major phases. Moreover, when emulating the automatic operating mode of the flight instrument, the RLS ExoMars Simulator also detected several minor compounds (pyroxene and brucite), some of which were not observed by NIR and XRD (e.g., calcite). Thereafter, we produced RLS-dedicated calibration curves (R2 between 0.9993 and 0.9995 with an uncertainty between ±3.0% and ±5.2% with a confidence interval of 95%) to estimate the relative content of olivine and serpentine in the samples. Our results show that RLS can be very effective in identifying serpentine, a scientific target of primary importance for the potential detection of biosignatures on Mars-the main objective of the ExoMars rover mission.

ExoMars Raman Laser Spectrometer (RLS): development of chemometric tools to classify ultramafic igneous rocks on Mars.

This work aims to evaluate whether the multi-point analysis the ExoMars Raman Laser Spectrometer (RLS) will perform on powdered samples could serve to classify ultramafic rocks on Mars. To do so, the RLS ExoMars Simulator was used to study terrestrial analogues of Martian peridotites and pyroxenites by applying the operational constraints of the Raman spectrometer onboard the Rosalind Franklin rover. Besides qualitative analysis, RLS-dedicated calibration curves have been built to estimate the relative content of olivine and pyroxenes in the samples. These semi-quantitative results, combined with a rough estimate of the concentration ratio between clino- and ortho-pyroxene mineral phases, were used to classify the terrestrial analogues. XRD data were finally employed as reference to validate Raman results. As this preliminary work suggests, ultramafic rocks on Mars could be effectively classified through the chemometric analysis of RLS data sets. After optimization, the proposed chemometric tools could be applied to the study of the volcanic geological areas detected at the ExoMars landing site (Oxia Planum), whose mineralogical composition and geological evolution have not been fully understood.

Detection of Potential Lipid Biomarkers in Oxidative Environments by Raman Spectroscopy and Implications for the ExoMars 2020-Raman Laser Spectrometer Instrument Performance.

The aim of the European Space Agency's ExoMars rover mission is to search for potential traces of present or past life in the swallow subsurface (2 m depth) of Mars. The ExoMars rover mission relies on a suite of analytical instruments envisioned to identify organic compounds with biological value (biomarkers) associated with a mineralogical matrix in a highly oxidative environment. We investigated the feasibility of detecting basic organics (linear and branched lipid molecules) with Raman laser spectroscopy, an instrument onboard the ExoMars rover, when exposed to oxidant conditions. We compared the detectability of six lipid molecules (alkanes, alkanols, fatty acid, and isoprenoid) before and after an oxidation treatment (15 days with hydrogen peroxide), with and without mineral matrix support (amorphous silica rich vs. iron rich). Raman and infrared spectrometry was combined with gas chromatography-mass spectrometry to determine detection limits and technical constraints. We observed different spectral responses to degradation depending on the lipid molecule and mineral substrate, with the silica-rich material showing better preservation of organic signals. These findings will contribute to the interpretation of Raman laser spectroscopy results on cores from the ExoMars rover landing site, the hydrated silica-enriched delta fan on Cogoon Vallis (Oxia Planum).

ExoMars Raman Laser Spectrometer: A Tool for the Potential Recognition of Wet-Target Craters on Mars.

In the present work, near-infrared, laser-induced breakdown spectroscopy, Raman, and X-ray diffractometer techniques have been complementarily used to carry out a comprehensive characterization of a terrestrial analogue selected from the Chesapeake Bay impact structure (CBIS). The obtained data clearly highlight the key role of Raman spectroscopy in the detection of minor and trace compounds, through which inferences about geological processes occurred in the CBIS can be extrapolated. Beside the use of commercial systems, further Raman analyses were performed by the Raman laser spectrometer (RLS) ExoMars Simulator. This instrument represents the most reliable tool to effectively predict the scientific capabilities of the ExoMars/Raman system that will be deployed on Mars in 2021. By emulating the analytical procedures and operational restrictions established by the ExoMars mission rover design, it was proved that the RLS ExoMars Simulator can detect the amorphization of quartz, which constitutes an analytical clue of the impact origin of craters. Beside amorphized minerals, the detection of barite and siderite, compounds crystallizing under hydrothermal conditions, helps indirectly to confirm the presence of water in impact targets. Furthermore, the RLS ExoMars Simulator capability of performing smart molecular mappings was successfully evaluated.

A Raman spectral study of stream waters and efflorescent salts in Rio Tinto, Spain.

Acidic waters and sulfate-rich precipitates are found in mine tailings such as Rio Tinto (Huelva, SW, Spain). In this work we have characterized the chemical constituents of stream water and have identified some efflorescent salts and precipitates by means of Raman spectroscopy. Variable amounts of sulfate and bisulfate are found in the aqueous samples, suggesting changes in the acidity of the solutions. An estimation of the sulfate/water relative abundance is also given. Solid samples are readily identified as gypsum and as mixtures of hydrated hydroxysulfates belonging to the copiapite group. These results are consistent with previous works reporting the mineralogy and water composition of acid mine drainage-related sites, and proves the importance of Raman spectroscopy as a tool for accurate and noninvasive analyses of acid waters and associated geochemistry.

Acute retinal toxicity associated with a mixture of perfluorooctane and perfluorohexyloctane: failure of another indirect cytotoxicity analysis.

AIMS: To report new information related to acute retinal toxicity of Bio Octane Plus, a mixture of 90% perfluorooctane (PFO) and 10% perfluorohexyloctane. METHODS: This retrospective, descriptive case series reports the occurrence of acute retinal toxicity after vitreoretinal surgery in which Bio Octane Plus (batch number 1605148) was used as an endotamponade. Cytotoxicity biocompatibility tests and chemical analyses by Fourier-transformed infrared (FTIR) spectroscopy and gas chromatography-mass spectrometry (GC-MS) of the presumed toxic product were performed. RESULTS: Four patients presented with acute severe visual loss after uneventful ocular surgery assisted by Bio Octane Plus (batch number 1605148) as endotamponade. Patients experienced extensive retinal vascular occlusion leading to retinal and optic nerve atrophy. The viability of ARPE-19 cells directly exposed to the suspect batch for 30 min was 0%. The agarose overlay method used by the manufacturer according to European Union regulations and International Organization for Standardization (ISO) International Standards failed to detect toxicity. FTIR spectroscopy showed small differences between the non-toxic and toxic batches. GC-MS analysis showed the presence of bromotributyl stannane (whose toxicity was demonstrated in the dose-response curve) only in the toxic batch of Bio Octane Plus. CONCLUSION: This is the third report of retinotoxicity due to PFO in 4 years. The clinical profiles may be missed as they resemble other postsurgical complications; therefore, more cases worldwide could have gone unreported. Protocols to determine cytotoxicity of intraocular medical devices and approved by the ISO International Standards based on indirect methods have failed and should be revised to ensure safety.

A Polyextreme Hydrothermal System Controlled by Iron: The Case of Dallol at the Afar Triangle.

One of the latest volcanic features of the Erta Ale range at the Afar Triangle (NE Ethiopia) has created a polyextreme hydrothermal system located at the Danakil depression on top of a protovolcano known as the dome of Dallol. The interaction of the underlying basaltic magma with the evaporitic salts of the Danakil depression has generated a unique, high-temperature (108 °C), hypersaline (NaCl supersaturated), hyperacidic (pH values from 0.1 to -1.7), oxygen-free hydrothermal site containing up to 150 g/L of iron. We find that the colorful brine pools and mineral patterns of Dallol derive from the slow oxygen diffusion and progressive oxidation of the dissolved ferrous iron, the iron-chlorine/-sulfate complexation, and the evaporation. These inorganic processes induce the precipitation of nanoscale jarosite-group minerals and iron(III)-oxyhydroxides over a vast deposition of halite displaying complex architectures. Our results suggest that life, if present under such conditions, does not play a dominant role in the geochemical cycling and mineral precipitation at Dallol as opposed to other hydrothermal sites. Dallol, a hydrothermal system controlled by iron, is a present-day laboratory for studying the precipitation and progressive oxidation of iron minerals, relevant for geochemical processes occurring at early Earth and Martian environments.

Spectroscopic study of olivine-bearing rocks and its relevance to the ExoMars rover mission.

We present the compositional analysis of three terrestrial analogues of Martian olivine-bearing rocks derived from both laboratory and flight-derived analytical instruments. In the first step, state-of-the-art spectroscopic (XRF, NIR and Raman) and diffractometric (XRD) laboratory systems were complementary used. Besides providing a detailed mineralogical and geochemical characterization of the samples, results comparison shed light on the advantages ensured by the combined use of Raman and NIR techniques, being these the spectroscopic instruments that will soon deploy (2021) on Mars as part of the ExoMars/ESA rover payload. In order to extrapolate valuable indicators of the mineralogical data that could derive from the ExoMars/Raman Laser Spectrometer (RLS), laboratory results were then compared with the molecular data gathered through the RLS ExoMars Simulator. Beside correctly identifying all major phases (feldspar, pyroxene and olivine), the RLS ExoMars Simulator confirmed the presence of additional minor compounds (i.e. hematite and apatite) that were not detected by complementary techniques. Furthermore, concerning the in-depth study of olivine grains, the RLS ExoMars simulator was able to effectively detect the shifting of the characteristic double peak around 820 and 850 cm-1, from which the FeMg content of the analyzed crystals can be extrapolated. Considering that olivine is one of the main mineral phases of the ExoMars landing site (Oxia Planum), this study suggests that the ExoMars/RLS system has the potential to provide detailed information about the elemental composition of olivine on Mars.

Raman signal processing software for automated identification of mineral phases and biosignatures on Mars.

Data from the ESA ExoMars Rover Mission will provide invaluable input for further studies in astro/exobiology. The search for mineral products as indicators of present and/or past biogenetic activities in Mars' surface and subsurface samples is the main objective of the compact Raman-laser-induced breakdown spectroscopy (LIBS) instrument. The inherent features of Raman spectroscopy and LIBS make the combined instrument a unique and very powerful tool in the search for biomarkers and hence it is regarded as the highest priority instrument for mineral analysis within the mission. We have developed a software package for the on-board processing of the instrument's data outputs, including spectral conditioning and search-match characterization of mineral phases and biomarkers. In this paper we show the mathematical and physical basis of the software package.

Comparison between direct contact and extract exposure methods for PFO cytotoxicity evaluation.

A series of recent acute blindness cases following non-complicated retinal detachment surgery caused the release of several health alerts in Spain. The blindness was attributed to certain lots of perfluoro-octane (PFO; a volatile and transient medical device). Similar cases have been reported in other countries. This has raised questions regarding the validity of cytotoxicity test methods currently used to certify the safety of PFO lots. The tests were performed according to the International Organization for Standardization (ISO) norms, using the extract dilution method or the indirect contact method as applied to L929 cells, a line derived from mouse fibroblasts. The limitations of those methods have been resolved in this study by proposing a new cytotoxicity test method for volatile substances. The new method requires direct contact of the tested substance with cells that are similar to those exposed to the substance in the clinical setting. This approach includes a few new technical steps that are crucial for detecting cytotoxicity. Our new method detected toxic PFO lots that corresponded to the lots producing clinical blindness, which previous methods failed to detect. The study suggests applying this new method to avoid occurrence of such cases of blindness.

Sulphate efflorescent minerals from El Jaroso Ravine, Sierra Almagrera--An SEM and Raman spectroscopic study.

Two sulphate efflorescent evaporite mineral samples from Jaroso, Spain have been studied by scanning electron microscopy and Raman spectroscopy. SEM by comparison with known minerals shows the evaporite mineral is a mixture of halotrichite and jarosite, whilst the oxidised mineral is predominantly jarosite. SEM characterises the halotrichite as long narrow crystals and the jarosite as distorted rhombohedral crystals. Raman spectra of the sulphates of K, Mg, Fe(II), Fe(III) are compared with the spectra of halotrichite, jarosite and the two sulphate efflorescent samples. The efflorescent sample was proven by Raman spectroscopy to be a mixture of halotrichite and jarosite and the oxidised efflorescent sample to be jarosite and a complex mixture of sulphates.

Habitability on Early Mars and the Search for Biosignatures with the ExoMars Rover.

The second ExoMars mission will be launched in 2020 to target an ancient location interpreted to have strong potential for past habitability and for preserving physical and chemical biosignatures (as well as abiotic/prebiotic organics). The mission will deliver a lander with instruments for atmospheric and geophysical investigations and a rover tasked with searching for signs of extinct life. The ExoMars rover will be equipped with a drill to collect material from outcrops and at depth down to 2 m. This subsurface sampling capability will provide the best chance yet to gain access to chemical biosignatures. Using the powerful Pasteur payload instruments, the ExoMars science team will conduct a holistic search for traces of life and seek corroborating geological context information. Key Words: Biosignatures-ExoMars-Landing sites-Mars rover-Search for life. Astrobiology 17, 471-510.