Effects of Receiving-Water Quality and Wastewater Treatment on Injury, Survival, and Regrowth of Fecal-Indicator Bacteria and Implications for Assessment of Recreational Water Quality By Donna S. Francy, Teresa L. Hart, and Cathy M. Virosteck U.S. Geological Survey Water-Resources Investigations Report 96-4199 Prepared in cooperation with Ohio Water Development Authority, Northeast Ohio Regional Sewer District, Summit County Columbus, Ohio 1996 Previous Studies Considerable information is available from laboratory studies showing that fecal-indicator bacteria surviving chlorination are able to repair injuries and survive in the aquatic environment. Investigators determined that the proportion of bacteria surviving chlorination increased when chlorinated wastewater was diluted with streamwater or distilled water (Heukelekian, 1951), as chlorine-contact time decreased (Braswell and Hoadley, 1974), or as residual chlorine concentration decreased (Shuval and others, 1973). In another study (Kinney and others, 1978), investigators found that the concentration of coliform bacteria in unchlorinated wastewater effluent, including those in the fecal-coliform group, steadily declined during a 5-day study, whereas the concentration of coliforms in the same effluent increased after chlorination. Because of the natural die-off pattern noted in the unchlorinated wastewater, the authors attributed the increase in bacteria concentration in chlorinated wastewater to repair of injured organisms rather than growth of healthy organisms. It was also suggested that standard selective MF methods for enumerating fecal-indicator bacteria do not support the growth of chlorine-injured bacteria (Kinney and others, 1978). Standard methods, defined as those methods believed to represent the best current practice of American water analysts, are generally applicable to ordinary problems of sanitary investigations (American Public Health Association and others, 1992). Selective methods are defined as those methods that inhibit growth of nontarget bacteria groups in order to encourage growth of target organisms. The most probable number (MPN) method, containing a nonselective enrichment step, was superior to the standard selective MF method in parallel tests for enumerating chlorine-injured fecal coliforms (Lin, 1973) and in parallel tests for enumerating unchlorinated and chlorine-injured coliforms (Mowat, 1976). The USEPA (U.S. Environmental Protection Agency, 1978) recommends that the standard selective MF method should not be used with chlorinated wastewaters and that any decision to use this test requires parallel MF/MPN evaluations to determine applicability of the MF method. However, because the MPN method is time-consuming, cumbersome, and an estimate of the "most probable number," many investigators sought improvements to the standard-selective MF method that would enhance growth of chlorine-injured bacteria (Camper and McFeters, 1979). Many enhanced-recovery MF methods have a pre-enrichment step on nonselective media, a temperature acclimation time, and (or) an alternate media devoid of inhibitory chemicals. Several investigators used standard-selective MF methods and (or) MPN methods to investigate survival of chlorine-injured bacteria in the field. Silvey and others (1974) determined concentrations of total coliforms and fecal coliforms by use of MPN methods in the Trinity River in Texas, downstream from four wastewater treatment plants. The investigators suggested that nonfecal-coliform strains exhibited significant regrowth following chlorination, whereas fecal-coliform strains failed to regrow in the river. In a study in the Chicago area (Haas and others, 1988), investigators used standard selective MF methods to compare concentrations of fecal coliforms in receiving waters before and after a wastewater treatment plant discontinued chlorination. They suggested that, beyond a certain affected zone, chlorination of an effluent may not improve microbiological water quality in the receiving stream. However, these studies were not done by use of a controlled group of fecal-indicator bacteria; and contributing influences from surface-water runoff and combined-sewer overflows could not be ruled out. Bissonnette and others (1975) investigated the influence of environmental stress on Escherichia coli (E. coli) injury and survival by use of membrane-filter chambers containing a controlled population of bacteria. E. coli is a major species in the fecal-coliform group and a natural inhabitant of the intestinal tract of warm-blooded animals. The investigators observed that, upon exposure to the aquatic environment, concentrations of E. coli obtained by use of a selective method were less than those obtained by use of an enhanced-recovery method. They also observed substantial variation in percentage of injury and survival of E. coli in various stream environments. Information regarding the effects of ambient water-quality conditions and wastewater-plant chlorination practices on injury and survival of fecal-indicator bacteria in receiving waters is lacking. However, the survival of chlorine-injured fecal-indicator bacteria may have implications from a public-health and water-quality perspective. Fecal-indicator bacteria that survive but are injured from chlorination may be able to repair their injuries and regrow, provided that a suitable growth medium is available. That growth medium may be enhanced-recovery medium or the gastrointestinal tract of an unsuspecting swimmer. Standard selective methods may not detect injured organisms; therefore, the enumeration of fecal-indicator bacteria and the pathogenic bacteria whose presence they indicate may be underestimated. Therefore, determining the health risk of swimming in receiving waters is difficult if information on enhanced-recovery method concentrations is not available. Wastewater treatment affects the injury, survival, and regrowth of fecal-indicator bacteria; http://oh.water.usgs.gov/reports/111/rpt96.4199.html#HDR0