HORTSCIENCE VOL. 41(1) FEBRUARY 2006 108 HORTSCIENCE 41(1):108–112. 2006. Received for publication 15 Aug. 2005. Accepted for publication 23 Sept. 2005. This research was supported though a grant from NASA’s Office of Biological and Physical Research Programs (NNK04EB08A) and the Life Sciences Service Contract at (NAS10-02001) at NASA’s Kennedy Space Center. Support for R. Hickey was provided through a Graduate Training Internship from FÁS (Foras Áiseanna Saothair), Ireland’s Training and Employment Authority. The authors also wish to acknowledge the support of Oscar Monje and Jessica Prenger technical discussion and software development. 1 To whom reprint requests should be sent; e-mail stuttgw@kscems.ksc.nasa.gov. Bioactivity of Volatile Alcohols on the Germination and Growth of Radish Seedlings G.W. Stutte, 1 I. Eraso, and S. Anderson Dynamac Corporation, Space Life Sciences Lab, Kennedy Space Center FL, 32899 R.D. Hickey University College Cork, Cork, IE Additional index words. VOCs, ethanol, methanol, 2-propanol, t-butanol, air pollution, radish, SMAC Abstract. A series of experiments were conducted to determine the sensitivity of radish to four light alcohols (ethanol, methanol, 2-propanol, and t-butanol) identified as atmospheric contaminants on manned spacecraft. Radish (Raphanus sativus L. ‘Cherry Bomb’ Hybrid II) seedlings were exposed for 5 days to concentrations of 0, 50, 100, 175, 250, and 500 ppm of each alcohol and the effect on seedling growth was used to establish preliminary threshold response values. Results show a general response-pattern for the four alcohol exposures at threshold responses of 10% (T 10 ), 50% (T 50 ) and 90% (T 90 ) reduction in seed- ling length. There were differences in the response of seedlings to the four alcohols, with the T 10 for t-butanol and ethanol (25 to 40 ppm) being 3 to 5× lower than for methanol or 2-propanol (110 to 120 ppm). Ethanol and t-butanol exhibited similar T 50 values (150 to 160 ppm). In contrast, T 50 for methanol (285 ppm) and 2-propanol (260 ppm) were about 100 ppm higher than for ethanol or t-butanol. Chronic exposures to 400 ppm t-butanol, ethanol or 2-propanol were highly toxic to the plants. Radish was more tolerant of metha- nol, with T 90 of 465 ppm. Seeds did not germinate at the 500 ppm treatment of t-butanol, 2-propanol, or ethanol. There were significant differences in projected performance of plants in different environments, dependent upon the regulatory guidelines used. The use of exposure guidelines for humans is not applicable to plant systems. Accumulation of air pollutants present in the atmosphere of closed environments such as the Space Shuttle, International Space Sta- tion (ISS), or proposed lunar or planetary bases are a concern to mission planners because of the potential effects on human health and the vehicle life support systems. Chronic exposure to volatile organic compounds (VOCs) in open systems, such as greenhouses or laboratories, which are in proximity to industrial sites, may also reduce plant yield and crop development [Sharkey, 1991; U.S. Environmental Protection Agency (EPA), 1978] Threshold exposure values (TEVs) for atmospheric contaminants are recommended by the American Conference of Governmental Industrial Hygienists (ACGIH, 2005) and the U.S. Department of Labor (DOL) Occupational Safety and Health Administration (OSHA) have established personnel exposure limits (PELs) for regulatory purposes (DOL, 1988). These industrial standards are based on the continuous exposure limit a typical worker could tolerate during a typical 8-h day (40-h work week) without suffering negative health effects. The National Aeronautic and Space Admin- istration (NASA) developed 1 h, 24 h, 7 d, 30 d, and 180 d spacecraft maximum allowable concentration (SMAC) values for individual trace chemical contaminants to ensure the health, safety, and performance of the crew members during space missions [National Research Coun- cil (NRC), 1994, 1996a, 1996b, 2000]. These values take into account a number of unique factors associated with space flight, including uniform good health of the crew and absence of pregnant or very young individuals (NRC, 1992), and are generally more conservative than either the ACGIH and OSHA values. Experiments were undertaken as a part of NASA’s Advanced Life Support Program to determine whether VOCs accumulate in closed plant chambers and what, if any, impact they had on plant growth. A large (113-m 3 ),closed plant growth facility at Kennedy Space Center (KSC), the biomass production chamber (BPC), permitted the tracking of VOC production and accumulation through growth and development of a number of crops (Batten et al., 1993, 1995, 1996; Stutte, 1999; Stutte and Wheeler, 1997; Wheeler et al., 1996b). During those tests, ethylene was the VOC with the greatest bioactivity and has generated the greatest amount of attention with respect to monitoring and control in spacecraft (Abeles, 1992; Klassen and Bugbee, 2004; Salibury et al., 1997; Wheeler et al., 2004). Although ethylene has received the greatest research attention, it is by no means the only biologically active VOC that accumulates. Batten et al. (1993, 1995, 1996) identified >170 VOCs in the BPC atmosphere from sev- eral crop species. The compounds originated from both anthropogenic (hardware, adhesives, equipment) and biogenic (plants, microorgan- isms) sources (Batten et al., 1993, 1996; Stutte and Wheeler, 1997). The production of biogenic VOCs varied depending on crop and stage of de- velopment (Batten et al. 1995; Stutte 1999). Periodic sampling of the atmosphere of the Space Shuttle, ISS, and other spacecraft has revealed the presence of trace concentrations of several additional VOCs (James et al, 1994; NASA, 2004; Perry 1998). Some of the most common VOCs that accumulate in spacecraft are light alcohols (ethanol, methanol, 2-propanol, and t-butanol) that originate from a variety of anthropogenic and biogenic sources (Perry, 1998; Perry and Peterson, 2003). Preliminary tests revealed that existing NASA SMAC levels to insure crew health were not sufficient to insure the health of a crop (Eraso et al., 2001, 2003; Stutte et al., 2004). For example, ethylene, one of the most problematic biogenic VOCs for plants, was found to have a threshold response in radish at 25 to 50 ppb (Eraso et al., 2003; Klassen and Bugbee, 2004) a concentration 40,000× lower than the human SMAC value. The lack of ethylene removal capabilities onboard some spacecraft has resulted in the accumulation of ethylene to >1 ppm and was implicated as primary cause of lack of seed development of wheat grown onboard the Russian Space Station MIR (Salisbury et al., 1997). The bioactivity of spacecraft VOCs other than ethylene has also been examined. Stutte et al. (2004a, 2004b) reported that chronic ex- posure of three radish cultivars (‘Sora’, ‘Cherry Belle’, and ‘Cherry Bomb’Hybrid II) to ethanol concentrations at 500 ppm (one-half the levels established NASASMAC, OSHAPEL, andAC- GIH TLV) resulted in severe stunting, reduced leaf area, and inhibition of hypocotyl expansion for all cultivars. At 100 ppm (10% of crew and worker exposure guidelines) the harvest index, leaf area index and absolute growth rate of the three cultivars was reduced. A seedling bioassay system was designed in order to rapidly screen VOCs for bioactiv- ity to prioritize compounds for further whole plant testing (Stutte et al., 2005). These experi- ments utilized the radish seedling bioassay to establish preliminary threshold, sensitivity, and lethal concentrations for the bioactivity of light alcohols in order to establish reasonable exposure guidelines for plant systems to sup- port long duration space systems (Stutte, 1999; Stutte et al., 2005). Materials and Methods Plant material and growth. Raphanus sativus L. ‘Cherry Bomb’ (Burpee Seed, Warminster, Pa.) was used in these experi-