United States Environmental Protection Agency Municipal Environmental Research Laboratory Cincinnati OH 45268 V f" . ./t" X Research and Development EPA-600/S2-84-116 Sept. 1984 &ER& Project Summary Evaluation of Urban Runoff and Combined Sewer Overflow Mutagenicity Stuart J. Spiegel, Edwin C. Tifft, Jr., Cornelius B. Murphy, Jr., and Randy R. Ott A study was conducted to evaluate combined sewer overflows and urban runoff for the presence of chemical mutagens. The Ames Salmonella/m\- crosome mutagenicity test was used as a general biological effects test for the qualitative detection of mutagens in the sanitary environment, including rain, urban runoff, sanitary wastewater, combined sewer overflows, sewage treatment plant effluent, and receiving waters. The Ames test is a relatively sensitive and simple bacterial test for detecting chemical mutagens. Its ad- vantages over long-term animal tests are speed, ease, and relatively low cost. The test employs previously mutated Salmonella typhimurium LT2 bacterial strains that tend to mutate back to their natural state when exposed to muta- genic compounds. Nineteen samples produced a detect- able response to one or more of the five S. typhimurium test strains, with or without metabolic activation. Nine of these samples (47%) were of urban runoff in the project area of metropoli- tan Syracuse (Onondaga County), New York, and they produced 17 of 30 detectable responses (57%). Five of the samples (26%) were from combined sewer overflows, and they produced 7 of 30 detectable responses (23%). The results indicated that urban runoff components that produce a detectable response in the Ames test may be diluted or inactivated in combi- nation with sanitary sewage to form combined sewage, since fewer responses were detected in the latter than in urban runoff. This Project Summary was developed by EPA's Municipal Environmental Research Laboratory, Cincinnati, OH. to announce key findings of the research project that is fully documented in a separate report of the same title (see Project Report ordering information at back). Introduction Only since the early 1960's have urban runoff and combined sewer overflows (CSO) been identified as pollutant contri- butors to receiving streams. And not until nearly 10 years later were urban runoff and CSO implicated as major sources of particulates, nutrients, and microbial loading to urban waterways. In recent years, almost any potential pollutant to be found in the urban environment can be traced to receiving streams via this pathway. To decrease surface water pollution from these sources, the U.S. Environ- mental Protection Agency (EPA) has engaged studies and provided funds for the design and construction of prototype CSO treatment facilities. These facilities are primarily designed to use some form of solids removal followed by disinfection. Though past studies have characterized urban runoff and CSO, and identified constituent pollutants, they have not con- clusively dealt with the most recent concerns about the fates and effects of organic chemicals in these systems. Potentially toxic, mutagenic, carcinogenic, or teratogenic chemicals are readily identifiable in the urban environment, but little attempt has been made to determine their concentrations in urban runoff and ------- Two alternative sites were also chosen m in case difficulties arose at any time in the collection of samples from the primary sites: (a) Site 004 - a large basin drainage area characterized by mixed residential and commercial land use; and (b) Site 074 - a drainage area with predominantly commercial and in- dustrial land use. In addition, three CSO samples were collected in Washington, D.C., one at each of three separate sites. Because of difficulties in obtaining manual grab samples at these sites, an automatic sampler was used. Also, two CSO samples were taken from a site in Rochester, New York (Site 007, Maple- wood Avenue), during two storm events. The sample collection sites were chosen using three selection criteria: 1. Uniqueness of drainage area char- acteristics. Drainage areas with percent imperviousness, popula- tion density, size, and land use mix representative of several others in the city were evaluated for selec- tion. In addition, drainage areas that were unique in the city (i.e., predominantly open space, such as Site 046) were selected. - 2. Ease and safety of sample col lee- fl tion. Where several sites of similar physical characteristics could have been selected, ease of sample collection was considered to pre- serve sample integrity. In addition, both receiving streams traverse busy urban areas where sample collection would be performed through manholes in streets. To ensure the safety of personnel, accessibility during high-traffic periods was considered. 3. Availability of baseline data. Phy- sical and chemical analyses of CSO's in the Syracuse area have been performed during previous characterization studies. These data could serve as a baseline for this study. This criterion was considered least important of the three in site selection. CSO samples were collected during four wet-weather events in glass con- tainers lowered from manholes directly above the overflow pipe. Sufficient sample was transferred to fill an amber glass bottle for Ames testing, a 50-ml plastic container with nitric acid preser- vative for TOC and heavy metals analysis, and a plastic half gallon container for other chemical/physical analyses. This sample storage and preservation proce- dure was followed for all samples. Glass CSO. Several reasons exist for this omission. Such studies would require the analysis of long lists of compounds, as in priority pollutant analysis, and these lists are constantly expanding. Technological difficulties exist in the analysis of such compounds. The detection limits of available instrumentation may not be sufficiently low to detect minute concen- trations of contaminants at levels that may still pose a potential health hazard. The cost of analyzing a single sample under these conditions is high, and the cost would be staggering to examine enough samples to provide statistically significant data for a long list of compounds. Furthermore, such determinations would still hot provide any biohazard risk index; that is, any measure of the biological effects these compounds might have in their environmental medium, particularly effects on the human system or synergis- tic or multiplicative effects. At this time, most treatment systems for CSO are still in the initial planning or evaluation phases. Though it is generally agreed that treatment should consist of solids removal and disinfection, there has been little evaluation of the need for additional levels of treatment. Because of the highly variable nature and occurrence of urban runoff and CSO, it is doubtful that the need for advanced treatment can be determined solely by characterization of a list of specific chemicals. Instead, general information describing the po- tential biohazard risk of these systems would be a valuable criterion for deter- mining the need for advanced treatment. This study has thus been based on the premise that urban runoff may substan- tially contribute substances of potential risk to the human environment and that a preliminary analytical survey using the Ames Salmonella/microsome mutagen- icity test might outline the presence and scope of that potential. To this end, samples were taken from seven sources of environmental contributors to the Onondaga County (Syracuse), New York, CSO system. These sources included: rain, urban runoff, dry-weather sanitary flows, and CSO, as well as influent and effluent from the Metropolitan Syracuse Treatment Plant (Metro) and samples from Onondaga Lake, the ultimate recep- tor of the overflows. Samples were tested by the Ames test and subjected to analyses for other chemical parameters, including total suspended solids (TSS), total organic carbon (TOC), 5-day bio- chemical oxygen demand (BOD5), oil and grease (O&G), and the heavy metals cadmium (Cd), chromium (Cr), lead (Pb), and copper (Cu). The results of the Ames test were compared with the baseline chemical analyses to determine any correlations that might help identify the source of mutagenic activity. Since inorganics (especially metals) are removed in the sample extraction procedure, the primary purpose of heavy metal analysis is to characterize the samples and evaluate whether their concentrations were typical or atypical of the sample type. This study was completed in two phases. Phase I involved the collection and analysis of 74 samples during 1980. Based on the results of those analyses, modifications were made to some of the procedures. Phase II involved the collec- tion and analysis of an additional 11 samples in 1981 from those drainage areas that elicited detectable responses during Phase I. Sample Collection As mentioned earlier, provisions were made in the original study plan to collect seven types of samples in Onondaga County (Syracuse), New York (CSO, dry- weather sanitary flow, urban runoff, influent and effluent to Metro, Onondaga Lake, and rain). CSO Samples The project plan required the selection and collection of one CSO sample during four wet-weather events from each of six CSO discharges. The object was to select six drainage areas to be examined, each with divergent characteristics of size, land use, population density, and/or percent imperviousness. Selection was made based on previous studies performed in the Syracuse area to characterized CSO sites. The physical characteristics of the six CSO sites that were selected are sum- marized as follows: (a) Site 005 - a small drainage area with a high percentage of impervious surface (86%) and predominantly commercial land use (however, few historical chemical data were avail- able from previous studies to charac- terize this area); (b) Site 019 - a large drainage area with mixed land use; (c) Site 027 - the only drainage area that includes a significant amount of industrial land use; (d) Site 037- a drainage area with a high percentage of impervious surface and mixed land use; (e) Site 043 - a large, high-density residential area; and (f) Site 046 - the only drainage area that is primarily open space. ------- containers were used for the collection and storage of Ames samples, since it was unknown what effect plastic would have on the samples. Dry-Weather Sanitary Flow Samples Sanitary sewage samples were collected from the combined sewer either before or after a storm event, as appropriate to the collection schedule, in the same manner as the CSO samples. Urban Runoff Samples Urban runoff samples were collected from catchbasins in their corresponding CSO sites. Where necessary, curbside runoff was used. Influent and Affluent to Metro Six samples were collected from the Metro plant. These included one set of influent and corresponding effluent samples during storm flow, a similar pair of samples during dry-weather flow, and two influent samples collected during separate wet-weather flow periods. O no n dag a Lake Samples Samples were collected from the north and south basins of Onondaga Lake 9 m from the Lake's surface, the lower level of the epilimnon. Rain Samples Rain samples were collected at the O'Brien & Gere Syracuse office in 36-in. diameter stainless steel pans prerinsed with dichloromethane. These pans were placed away from buildings, cars, trees, and pavement to minimize contamination from extraneous sources. Rain from two pans was combined until a sufficient sample was collected for analytical pur- poses. The pH of this sample was taken immediately. Mutagenicity Testing Phase I Criteria used in this study for determin- ing a significant level of mutagenicity were recommended by EPA. The signifi- cance for each strain is determined by the formula MAR=E-C where MAR=mutagenic activity ratio E =the number of induced re- vertants C =the number of spontane- ous revertants on the day that the E revertants were induced c =the historical rate of spon- taneous reversions in the testing laboratory. MAR values were calculated for all samples with all five test strains, without and with S-9 liver activation. A negative MAR results when E is less than C for a given test strain on a particular day. A zero value is obtained when E equals C. And a positive value results when E is greater than C, although this positive value may only be a fraction if E is only slightly greater than C. A sample is considered positive or detectable if the MAR is 2.5 or greater. Table 1 summarizes only those samples and the particular strain(s) with a MAR of 2.5 or greater. The MAR values presented are for 100 /ul of sample concentrated 200 times (the highest concentration of any sample available in this study and therefore the most likely sample dosage to induce mutagenic activity), with 500//I of S-9 reaction mixture and 100//I of the appropriate test culture. Nineteen sam- ples induced a detectable response in one or more of the test strains by this criterion. Nine of these 19 positive samples (or 47%) were of urban runoff; seven were collected from Syracuse and two from Rochester, New York. Five of the 19 samples (or 26%) were CSO samples — three from Onondaga County and two from Washington, D.C. Nine of the various samples listed in Table 1 as inducing a positive MAR were mutagenic in TA1538 yet not in the more sensitive counterpart, TA98. Some indications are that this is not an unusual test result; TA1538 may show greater sensitivity than TA98 with some chemi- cals. No MAR of 2.5 or greater was obtained from analyses of the following samples: CSO from Sites 4 and 46; sanitary wastewater from Sites 37, 43, and 46; urban runoff from Sites 27 and 46; influent to the Metropolitan Treatment Plant; and CSO from Rochester, New York. The results of all applied dosages for samples that produced a detectable MAR (equal to or greater than 2.5) appear in Table 2. Three concentrations (2X, 20X, and 200X) were applied at a dosage of 100//I/plate at each concentration. Nine of the 19 samples that produced detect- able responses (a positive MAR) did so across the range of all three concentra- tions; six of these responses resulted from metabolic activation. Four of these nine samples produced a MAR of 2.5 or greater for all three concentrations. The four curves produced from the responses of these samples are generally horizontal with little slope, indicating that these samples did not show an increase in the mutagenic response with an increase in the amount of sample applied. Generally, a response that increases with dosage or concentration would be expected; but a response such as that shown by these samples is not unusual. Only sample number 95871 exhibited a logarithmic response curve. Of the 30 detectable responses (those from more than one test strain for six of 19 samples), 10 were from Site 004 (in three samples), and four were from Site 037 (also in three samples). A more interesting observation is that 17 responses of the total of 30 (57%) were from nine runoff samples (47% of the 19 samples from which detectable responses were obtained). CSO samples produced seven responses from five samples, and dry- weather sanitary samples elicited four responses from three samples. Thus, urban stormwater runoff may contribute potentially mutagenic substances to CSO and receiving waters. But since mutagen- ic activity is not as evident in the respective CSO samples as it is in the urban runoff, it is probable that the concentrations of mutagenic substances in the runoff are diluted below the detectable limits of this study as the runoff mixes with sanitary waste to form CSO. Phase II For this verification phase of the study, an additional 11 samples were collected in Syracuse in the late summer of 1981. These samples included two runoff and two CSO samples from both Sites 004 and 037, two samples of Metro influent, and a rain sample. One gallon of each sample was extracted and brought to a volume of 20 ml with dimethyl sulfoxide (DMSO), resulting in a concentrate of 189.25 times the original sample. Five dosages (500, 400, 300, 200, and 100//I) were applied. In two instances, the samples did elicit a detectable response. One sample was CSO from Site 004, and the other was Metro influent. The CSO sample (OBG No. 28524) had a MAR of 5.2 for tester strain TA 1538 at a dosage of 200 /ul; higher dosages resulted in a weaker response. The Metro influent had a MAR of 6.1 for TA1538 at the highest applied dosage of 500 /t/l; lower dosages did not result in a MAR greater than 2.4. ------- Table 1. Phase I Mutagenicity Results MAR* TA98 Sample Number 94492 95366 95368 95475 95476 95477 94491 95739 94493 94497 95745 95871 95873 95474 95369 94765 94766 94662 94663 City 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 4 4 Source 1 1 1 2 2 2 3 3 3 3 3 3 3 4 7 1 1 3 3 ID Location 19 27 43 4 19 27 4 4 19 37 37 37 43 2 . 24 34 7 7 CODE Descriptor 1 1 1 2 2 2 1 1 1 1 1 1 1 2 . 1 1 1 1 Without S-9 -t - - - - - - 3.0 - 2.7 - - . _ _ - - - - With S-9 2.9 . - - - - 3.3 2.6 - - - - _ - - - - MAR MAR MAR MAR TA100 TA1535 TA1537 TA1538 Without S-9 2.8 - - - - . - - - _ - 6.8 . _ _ - 76.2 - - With Without With Without With Without With S-9 S-9 S-9 S-9 S-9 S-9 S-9 . 72 9.1 2.7 3.9 11.2 - - - - - 5.1 4.1 2.6 3.4 2.5 4.0 4.8 2.6 - - - - _ 29 2.9 ----- - 10.0 6.1 2.6 4.4 4.2 - 4.5 3.8 *MAR - mutagenic activity ratio. t - No activity at 2.5 or above. Table 2. Detectable Responses from Phase I Analyses Sample Number 94492 94492 95366 95368 95475 95476 95477 95477 94491 94491 94491 94491 94491 95739 95739 95739 95739 94493 94497 95745 95871 95871 95873 95474 95369 94765 94766 94766 94662 94663 Organism TA98 TA100 TA 1538 TA 1538 TA 1538 TA 1538 TA100 TA 1538 TA98 TA 1537 TA 1537 TA 1538 TA 1538 TA98 TA98 TA 1537 TA 1537 TA 1535 TA98 TA 1538 TAWO TA100 TA 1538 TA 1538 TA 1538 TA 1538 TAWO TA 1537 TA 1538 TA 1538 S-9 Reaction Mixture fal) 500 - 500 500 500 500 500 500 500 500 - 500 500 - 500 - - 500 - 500 500 500 500 - - - - - 200-Xt 2.9 2.8 7.2 9.1 2.7 3.9 11.2 5.1 3.2 4.1 2.6 3.4 2.5 3.0 2.6 4.0 4.8 2.6 2.7 2.9 68. 29. 10. 6.1 2.6 4.4 16.2 4.2 4.5 3.8 MAR* 20X 0.5 0.3 3.2 6.0 2.8 4.0 0.1 4.6 0.2 2.5 0.9 2.8 0.7 1.1 0.6 3.2 1.9 -0.1 0.3 -0.7 12. 1.7 2.1 5.6 1.3 0.8 -0.5 -0.5 0.6 0.6 2X - 1.2 2.3 3.4 4.0 - 5.4 - -0.7 0.2 - - - 0.8 - - - - 1.7 - 6.5 - - - - - - * MAR = mutagenic activity ratio. t Concentrated 200 times. ------- Two samples produced toxicity; runoff from Site 004 (OBG No. 28885) with TA100 (no activation) and Metro influent (OBG No. 28888) with TA100 (with and without activation). The four aforementioned samples that produced positive or toxic responses were re-analyzed for verification. None of the analyses resulted in a MAR of 2.5 or greater at any applied dosage. Conclusions The following conclusions resulted from the project: 1. Nineteen samples out of 85 total samples (22%) may be interpreted to have induced a detectable mutagenic response from one or more of the test strains, with or without metabolic activation. Nine of these samples were of urban runoff. Some samples required a 200-fold concentration for detection of this activity. Of 30 detectable responses, 17 (57%) were found in urban runoff, and seven (23%) were found in CSO. 2. The samples with a detectable response did not appear to be correlated to levels of any of the chemical parameters measured or to any particular CSO site. The experi- mental design involved too few samples and chemical parameters to obtain correlations between detect- able mutagenic activity and chemical characteristics of samples or sample types (CSO, urban runoff, etc.). 3. Fewer responses were detected in CSO than in urban runoff. Thus substances present in urban runoff that produce a detectable response in the Ames test may be diluted or inactivated when the runoff is added to sanitary sewage to form combined sewage (CSO). 4. This study indicates the need for a more comprehensive survey of the subject. Definitive conclusions are difficult because of problems en- counterd in analytical methodology, sample toxicity to test bacteria, and the inherent chemical variability of CSO and urban runoff. 5. The spot test (preliminary screening) may not be usable as a preliminary step in analyzing polluted aquatic samples with the Ames Salmonella/ microsomemutagenicity test because of sample toxicity at ambient concen- trations. 6. Dichloromethane (methylene chlo- ride) may serve as both a disinfec- tant and an extractant. But if extrac- tion or concentration is not required in other aquatic environmental samples, a more suitable disinfec- tion procedure may be necessary. The full report was submitted in fulfillment of Cooperative Agreement No. CR-806640 by O'Brien & Gere Engineers, Inc., and by the Department of Drainage and Sanitation, Onondaga County, under the sponsorship of the U.S. Environmental Protection Agency. Stuart J. Spiegel. Edwin C. Tifft, Jr., and Cornelius B. Murphy, Jr. are with O'Brien and Gere Engineers, Inc., Syracuse, NY 13221; and Randy R. Ottis with the County of Onondaga, Department of Drainage and Sanitation, North Syracuse, NY 13212. Richard Field (see below) and Robert Turkeltaub are the EPA Project Officers. The complete report, entitled "Evaluation of Urban Runoff and Combined Sewer Overflow Mutagenicity." (Order No. PB 84-211 168; Cost: $14.50, subject to change) will be available only from: National Technical Information Service 5285 Port Royal Road Springfield, VA 22161 Telephone: 703-487-4650 The EPA Project Officer, Richard Field, can be contacted at: Storm and Combined Sewer Program Municipal Environmental Research Laboratory—Cincinnati U.S. Environmental Protection Agency Edison, NJ 08837 *USGPO: 1984-759-102-10671 ------- United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 Official Business Penalty for Private Use $300 Pb OuO U 6 E^y/l* KFbluu > HkUTtCTlON IU fr 060^4 ------- |