Tracking the short-term fate of some organic pollutants of the ISS Crew Latent Condensate (methanol, ethanol, and isopropanol) when included in the nutrient solution for rocket (Eruca sativa Mill.) hydroponic cultivation Simona Proietti, Walter Stefanoni, Stefano Moscatello, Emanuele Pallozzi, Lucia Grizzaffi, Ilaria Locantore, Carlo Calfapietra, Marco Lauteri, Alberto Battistelli. CNR – IBAF Istituto di Biologia Agroalimentare e Forestale, Porano (TR) - Italy Thales Alenia Space Italia Torino - Italy DAFNE, Università della Tuscia, via San Camillo De Lellis snc, Viterbo (VT) - Italy International Project: Inflatable greenhouse for space plant food production and life support. Italian Ministry of Foreign Affairs (MAE) 10 th Review Conference on Scientific and Technological Cooperation Agreement between Italy and USA Workshop on Bioregenerative Life Support Turin, May 2015

Water streams from space station can contains various organic contaminants During long term human space mission, water recovered from various streams should be used as the basic component of nutrient solutions for plant cultivation. Methanol, Ethanol, and Iso-propanol (MEI) are included in the list of Wastewater Contaminants in Crew Latent Condensate Figure 2. Relative organic compound percentages in latent condensate wastewater. 5x10 -3 % 8x10 -4 % 4x10 -4 % (Wastewater Contaminants in Crew Latent Condensate: Handford, 2004). For this end the quality of recovery water needs to be carefully evaluated since plants growth and plant food quality can be affected by the quality of water used

Methanol, Ethanol, and Iso-propanol (MEI) are selected to investigate the effects of relevant contaminants found in the ISS on plant growth and on plant physiology. Rocket plant (Eruca sativa Mill.) grown from seeds in a growth chamber for 4 weeks in a floating system Feeding of MEI were imposed on rocket seeds and on rocket plants

Do the rocket seeds germinate? When the ethanol concentration was decreased at 0,2% until the concentration found in the water condensate of ISS the germination rate of rocket seed is not affected. 8x10 -4 % = ethanol found in the water condensate of ISS Methanol and isopropanol do not affect rocket seeds germination also at high concentration (1%) Ethanol affects seeds germination only at high concentration (1% and 0,5%) With MEI

Are there a rapid and destructive effects on rocket plants? With MEI Gas exchange measurements and fluorescence of chlorophyll a using a Licor XT 6400 after feeding with 15mM of MEI Feeding Alcohols did not affected photosynthesis rate nor fluorescence emission. Gas exchanges were unaltered by alcohols also at higher concentration to respect the concentration found in the water streams of ISS.

MEI Are they taken up and accumulated by rocket plants? Methanol and Ethanol, but not isopropanol, were detected in leaves after feeding, but a low concentration. Leaf extracts from plants fed with MEI 1% were analyzed by Ion Chromatography ICS 5000 (Dionex ThermoFisher) Are they translocated? Are they accumulated? Are they absorbed? Standard Expected Isopropanol in leaf Methanol and Ethanol (and also Isopropanol, see later) are taken up by the rocket root and translocated to leaves, but do not accumulate to high concentration.

MEI in plant tissues Metabolism Re-emission in the atmosphere CO 2 (complete destruction) Metabolites What is the fate of MEI once they are inside the plant ? Accumulation (low) Accumulation Formaldehyde - Formic acid Acetaldehyde - Acetic acid Acetone

Feeding of 13 C enriched MEI caused a remarkably change in  13C in the respiratory CO 2 with a decreasing intensity with increasing alcohol molecular mass. This clearly indicates the uptake of alcoholic compounds from plants and a fast turn-over of labeled methanol and ethanol molecules as soon as they enter on the plant metabolism. M and E can be metabolized to enter respiratory metabolism: plants can contribute to degradation of M and E to CO 2. Methanol 15 mM Isotopic ratio   C Ethanol 15 mM Isotopic ratio   C Isopropanol 15 mM Isotopic ratio   C Labeling experiments were carried out to test if MEI enter into the respiratory metabolism using 13 C enriched MEI Feeding was carried out with a solution (15 mM) of each alcohols containing a small proportion of 13 C labeled molecules. The presence of 13 C was measured (  13C) on CO 2 released by leaf respiration, by IRMS.

MEI Are their metabolic products accumulated in plant tissues? Organic acids, as a product of alcohols metabolism in plants were analyzed by Dionex™ ICS-5000 from leaf extracts from plants fed with MEI. Isopropanol Acetone Ethanol AcetaldehydeAcetic acid Methanol FormaldehydeFormic acidCO 2 oxidation

No accumulation of metabolic products of methanol were detected by IC An increment of acetic acid in leaves was detected after Ethanol feeding. Accumulation of metabolic products of MEI in rocket leaves. Acetic acid in leaves

Methanol Ethanol Isopropanol Are they released, in the atmosphere? Are their metabolic producs released? The emission in the atmosphere of MEI and relevant metabolites were analyzed on line by Real-time trace gas analyzer solutions based on Proton-transfer-reaction mass spectrometry (PTR-MS - Ionicon).  Methanol and formaldehyde and formic acid from methanol oxidation  Ethanol and acetaldehyde and acetic acid from ethanol oxidation  Isopropanol and acetaldehyde and acetone from isopropanol oxidation Isopropanol Acetone Ethanol AcetaldehydeAcetic acid Methanol FormaldehydeFormic acidCO 2 oxidation

PTR-MS showed that Isopropyl alcohol was adsorbed by rocket plants, translocated and re-emitted by rocket leaves. Methanol was emitted by leaves formaldehyde and formic acid was not detected. After ethanol feeding acetaldehyde was the most abundant organic compound emitted trough stomata. Isopropyl alcohol was also metabolized in acetaldehyde and in acetone reemitted in the atmosphere. Isopropil alcohol

An array of measuring techniques should be used to track the fate of each organic compound when it enters in contact with a plant via the nutrient solution delivery system. Even similar molecules can have a very different fate when in contact with plants. Plant metabolism can destroy the pollutant or even produce other equally or even more dangerous molecules and even release them in the atmosphere. There is no general rule to be applied. Specific interaction plant/pollutant should be tested with reference to the growing conditions. An accurate definition of organic pollutants that can enter in a plant growing system in Bioregenerative Life Support system is necessary to ensure :  plant growing and productive performances.  food quality and security.  the potential treat of release in the system of the same or derived pollutants. Conclusions Perspectives