Microbial Characterization and Population Changes in Nonpotable Reclaimed Water Distribution Systems HODON RYU, ABSAR ALUM, AND MORTEZA ABBASZADEGAN* National Science Foundation Water Quality Center, Department of Civil and Environmental Engineering, Arizona State University, P.O. Box 875306, Tempe, Arizona 85287-5306 Changes in the microbial quality of nonpotable reclaimed water distribution systems in seven metropolitan areas of the southwestern United States were investigated by performing pathogen monitoring and bacterial growth. Water samples were collected from tertiary-treated effluents at point of entry and point of use in the distribution systems. The samples were analyzed for Cryptosporidium oocysts, Giardia cysts, enteroviruses, microbial indicators, and assimilable organic carbon (AOC). Cryptosporidium and Giardia were detected in 16% (12/77) and 43% (33/77) of nonpotable reclaimed water samples, whereas no infectious Cryptosporidium parvum were detected in any of the samples. No infectious enteroviruses were detected in any sample (0/27). At point of entry total coliform and fecal coliforms ranged from 0.7 to 280 and from 0 to 1.9 colony- forming units (cfu)/100 mL, respectively. Increases in the number of coliforms were observed as water passed through the reclaimed water distribution systems. However, no such increase in the number of coliphages was found. The chlorination practices at some utilities were not sufficient to inactivate coliforms and coliphages, but supplemental ultraviolet disinfection resulted in lower numbers of these microbial indicators. AOC levels decreased by 3-fold as water passed through the distribution systems, which inversely correlated with bacterial regrowth. Introduction Diminishing freshwater supplies and increasing municipal water demands in highly populated areas make water reuse a significant global concern. Historically, reclaimed water was used to irrigate crops (1). In the last 30 years, dramatic changes in the quantity and quality of reclaimed water produced by megacities have resulted in new application scenarios. The water reuse trend has shifted toward uncon- ventional uses such as urban irrigation, toilet and urinal flushing, commercial and industrial uses, and indirect potable reuse (2). Southwestern states of the United States such as Arizona, California, Nevada, and Texas have extensive water reuse programs (3). However, concerns about the microbial content of reclaimed water limit its applications. A wide variety of enteric microbial pathogens, including viruses, bacteria, and protozoan parasites, may be found in wastewater (4). Occurrence of microbial pathogens in reclaimed water and the related risks have been examined over the last several decades (5-9). Recently, reports of infectious Cryptosporidium parvum oocysts in reclaimed water plant effluents have been published (10, 11). However, microbial water quality and growth in reclaimed water distribution systems remains to be considered. Reclaimed water, even tertiary-treated, usually contains higher amounts of organic matter than drinking water, indicating that consumption of chlorine in the reclaimed distribution systems could occur rapidly, resulting in conditions con- ducive for bacterial growth. Moreover, concentrations of assimilable organic carbon (AOC), which serves as nutrients for bacterial growth, are much higher in reclaimed water than in drinking water. On the basis of these two factors (12), it is expected that high nutrient levels in combination with low chlorine residuals initiate bacterial growth in reclaimed water distribution systems. Many studies on bacterial growth in drinking water distribution systems have been reported (13-17); however, few studies on microbial surveillance have been performed in nonpotable reclaimed water distribution systems. No federal regulations for water reuse exist in the United States. Individual states are responsible for the development and implementation of water reuse criteria (2, 18). Reclaimed water samples from seven metropolitan areas in the south- western United States were analyzed for a variety of micro- organisms and chemical parameters. This study characterizes water quality in reclaimed water distribution systems by monitoring pathogens and investigating microbial growth. The results of this study will aid in building a database of common microbes and their occurrence to be used in revising guidelines and disinfection practices for reclaimed water. Experimental Section Site Selection and Sampling Strategy. On the basis of utility size, treatment processes, and water reuse application, seven nonpotable reclaimed water distribution systems were selected (Table 1). In each distribution system two sampling sites (point of entry, POE, and point of use, POU) were selected for periodic monitoring for water quality indicators. Water samples from all the sampling sites were collected between June 2002 and June 2003. Samples for Cryptosporidium oocysts, Giardia cysts, male-specific coliphages, somatic coliphages, total coliforms, fecal coliforms, and HPC bacteria were assayed on a bimonthly basis, whereas samples for enteroviruses were collected and assayed on a quarterly basis only at POU in each distribution system. Chemical parameters such as AOC, dissolved organic carbon (DOC), and total chlorine residuals were also measured quarterly. Sample Collection and Processing. The samples for protozoan parasites and enteroviruses were collected in a 100-L plastic container and dechlorinated by adding 500 mL of 10% sodium thiosulfate. The water samples for Crypto- sporidium and Giardia were filtered through an Envirochek- HV sampling capsule (Gelman Sciences, Ann Arbor, MI) at flow rates of no more than 2 L/min, and the samples for viruses were filtered through a 1 MDS filter (CUNO Inc., Meriden, CT) at 20 L/min. The water samples were continu- ously mixed during filtration process. Sample volumes for protozoan parasites and enteroviruses ranged from 20 to 170 L and from 114 to 341 L, respectively. Sample volumes varied due to differences in water turbidity at each utility. Filter cartridges retained in filter housings were shipped overnight at 4 °C to the Environmental Microbiology Laboratory at Arizona State University, Tempe, AZ. Samples for Cryptosporidium, Giardia, and enteric viruses were * Corresponding author phone: (480) 965-3868; fax: (480) 965- 0557; e-mail: abbaszadegan@asu.edu. Environ. Sci. Technol. 2005, 39, 8600-8605 8600 9 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 39, NO. 22, 2005 10.1021/es050607l CCC: $30.25  2005 American Chemical Society Published on Web 10/13/2005