ORIGINAL ARTICLE Environmental Inactivation of Cryptosporidium parvum Oocysts in Waste Stabilization Ponds Roberto Reinoso & Eloy Bécares Received: 25 September 2007 / Accepted: 11 February 2008 / Published online: 15 March 2008 # Springer Science + Business Media, LLC 2008 Abstract The survival of Cryptosporidium parvum oocysts in a waste stabilization pond system in northwestern Spain and the effects of sunlight and the depth and type of pond on oocyst viability were evaluated using an assay based on the exclusion or inclusion of two fluorogenic vital dyes, 4′ ,6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI). All tested factors had significant effects (P < 0.01) over time on C. parvum oocyst viability. Sunlight exposure was the most influential factor for oocyst inactivation. A 40% reduction was observed after 4 days exposure to sunlight conditions compared with dark conditions. The type of pond also caused a significant reduction in C. parvum oocyst viability (P <0.01). Inacti- vation rates reflected that the facultative pond was the most aggressive environment for oocysts placed both at the surface (presence of sunlight) and at the bottom (absence of sunlight) of the pond, followed by the maturation pond and the anaerobic pond. The mean inactivation rates of oocysts in the ponds ranged from 0.0159 to 0.3025 day −1 . Introduction The protozoan parasite Cryptosporidium parvum is a zoonotic pathogen capable of infecting the epithelial cells lining the digestive tract of numerous host species, including humans. Their oocysts, environmentally robust, have been responsible for numerous outbreaks of water- borne diseases worldwide causing serious implications for public health [16, 17, 35]. Numerous studies have evaluated the resistance of oocysts to disinfectants used in water and wastewater treatment plants, i.e., ozone, chlorine, chlorine dioxide, monochloramine, UV light [23, 24]. However, less attention has been directed to survival of C. parvum oocysts in natural aquatic environments where a wide range of environmental pressures are present, including climatic conditions [34]. Waste stabilization ponds (WSPs) have been widely used for biological treatment of domestic sewage being particu- larly efficient in the removal of pathogens [18, 38]. Previous studies have indicated that sunlight may be the main factor for the inactivation of microorganisms in these systems [8, 9], suggesting that disinfection models incor- porating sunlight are more satisfactory than those that do not [26]. Sunlight disinfection in WSPs may be limited by factors affecting the penetration of solar radiation into pond water such as depth [25], algal concentration [31], or mixing [28]. Furthermore, previous investigations have demonstrated that environmental variables such as pH, dissolved oxygen (DO), and humic substances may interact with sunlight modifying the mechanisms of radiation- induced damage on microorganisms [6]. Other factors widely demonstrated to be responsible for inactivation of C. parvum oocysts under laboratory con- ditions are temperature [12, 13, 15, 20], pH [3, 11], water potential [39], and ammonia [14, 21]. However, few studies have evaluated the effects of these factors on survival of oocysts under field conditions. Robertson et al. [33], using semi-permeable containers exposed oocysts under tap and river water environments demonstrating that C. parvum Microb Ecol (2008) 56:585–592 DOI 10.1007/s00248-008-9378-7 R. Reinoso (*) Environmental Research Institute, University of León, La Serna 58, 24007 León, Spain e-mail: rreit@unileon.es E. Bécares Department of Ecology, Faculty of Biological and Environmental Sciences, University of León, 24071 León, Spain