Mutagenic Activity in Organic Wastewater Concentrates Stephen M. Rappaport", Michael G. Richard, Monica C. Hollstein, and Ronald E. Talcott University of California, School of Public Health, Berkeley, Calif. 94720
Organic wastewater concentrates from six treatment plants were tested for mutagenicity using the Ames mutagen bioassay. Concentrates were prepared by passing 4-8 L of wastewater through two porous copolymer resins (Amberlite XAD-2 and XAD-7) in series. Of four tester strains, TA-98 in the presence of Arochlor-induced rat liver enzymes was the most sensitive indicator of mutagenicity. Mutagenic responses were observed in extracts from most of the plants tested. More activity was found in secondary and post-secondary extracts than in primary extracts. Dose-response curves obtained for selected samples showed a maximum activity of 210 revertants/mg of extract (TA-98 S-9). Fractionation of positive extracts showed the activity to be primarily in the basic and neutral fractions, while acid fractions were relatively inactive.
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The state of California faces predicted water shortages in the near future. Much effort is currently underway to upgrade existing wastewater treatment plants and to reuse reclaimed water where lower quality water can be used or to recycle municipal supplies by groundwater recharging. Technical advances in wastewater treatment processes, already in use in large scale operations, appear to be capable of meeting gross impurity requirements for reusable effluents. However, the health significance of organic materials that remain in water following treatment is still questioned. The purpose of the study reported herein was to determine the mutagenic activities of various wastewater effluents produced by advanced wastewater treatment methods. Much research has been performed on the types and amounts of organic materials in wastewaters and treated effluents. A varied list of compounds from both naturally occurring and anthropogenic sources has been reported (1-10). Much less is known about the delayed health effects of organic compounds in water. Ongerth et al. ( I 1 ), in a summary of the available literature in 1973, noted that most studies involving the administration of organic concentrates from water and wastewater to animals had not shown any carcinogenicity. Ottoboni and Greenberg indicated that two of ten male rats given filtered wastewater as drinking water developed tumors (12). This observation has neither been repeated nor extended, however. Page et al. (13) reported that there appeared to be significantly higher cancer mortality rates among Louisiana residents who obtained their drinking water from the Mississippi River rather than from underground sources. Pelon et al. ( 1 4 ) reported an observation of mutagenicity in water. The authors indicated that samples of unconcentrated Mississippi River water were mutagenic in histidinedependent strains of Salmonella typhzmurium (Ames test). Significant changes in methodology and interpretation of data from those reported by Ames et al. (15) make these observations equivocal, however. Mutagenicity, in this bioassay, should produce a t least twice the number of revertants as concurrent negative controls and should provide a clear dose-response relationship. Such criteria were not met in the study of Pelon et al. Loper et al. ( 1 6 ) tested concentrates from water supplies o f several cities in the United States using the Ames test. Mutagenesis was detected in samples from each city and the authors noted that the activity was observed in the absence of Arochlor-induced rat liver enzymes (S-9). Simmons et al. (17) tested 71 pure organic compounds which have been 0013-936X/79/0913-0957$01,00/0
identified in drinking wzter to determine if they were mutagenic in the Ames test. Thirty-four percent of these substances, primarily volatile alkyl halides (products of chlorine disinfection), were mutagenic.
Experimental Sampling. Five wastewater treatment plants were chosen for this study from the greater San Francisco Bay area (plants A-E) and one from the greater Los Angeles area (plant F). Descriptions of the treatment processes are given in Table I. Two criteria were applied for selection of the plants. First, each uses a post-secondary wastewater treatment process that produces an effluent suitable for reuse applications. Indeed, four of the plants are reusing their effluents. Second, the plants process incoming wastes from a variety of sources to include entirely domestic and mixed domestic-industrial wastes. The primary sedimentation process and the secondary treatment-activated sludge process are similar among the six treatment plants studied. Major differences in treatment occur during the post-secondary processes. One plant uses only simple chlorination, four use filtration through either carbon, anthracite, or sand, and two use flocculation. All use either chlorine or ozone disinfection with or without sulfur dioxide dechlorination. All plants using chlorine add enough t o yield a chlorine residual of from 3 to 12 mg/L prior to dechlorination. Grab samples were collected a t three sites in each treatment plant. The first set was obtained a t the outlet of the primary sedimentation basin, the second a t the secondary clarifier (prior to disinfection), and the third a t the discharge point after all treatment processes. Between 4 and 8 L of each sample was collected and transported in amber glass bottles. Samples were processed within 3 h of collection. Concentration of Organics. Various methods were reviewed for the extraction and concentration of organic materials from water and wastewater. These included solvent extraction (18, 19), reverse osmosis (20),low-temperature vacuum distillation (21), ion exchange (22), and adsorption by activated carbon (23) or Rohm and Haas Amberlite XAD resins (24-31). Of these, the use of XAD resins appeared to provide the most convenient and reproducible approach for isolating unknown organic substances from large volumes of water. The retention behavior of these resins has been well characterized by Pietrzyk and Chu (32,33), who showed that they all behave as reversed-phase chromatographic media with qualitative differences in retention of polar species among the resin types. A collection system employing a mixed bed of XAD-2 (nonpolar) and'XAD-7 (moderately polar) was selected in an attempt to ensure the collection of a t least a portion of most chemical species over a range of polarities. A similar concentration method has been reported (31). The resins were obtained from Mallinckrodt Chemical Co. as 20/50 mesh spherical particles. They were washed with water and Soxhlet extracted with 95% ethanol until no maxima were observed in UV-visible spectra of the washings and were stored in 95% ethanol prior to use. Glass columns (19 mm i.d. X 300 mm) were loaded with 35 em.
