United States Environmental Protection Agency Health Effects Research Laboratory Research Triangle Park NC 27711 Research and Development EPA-600/S1-83-012 Sept. 1983 &EPA Project Summary Enteric Virus Removal in Wastewater Treatment Lagoon Systems Howard T. Bausum, Stephen A. Schaub, William E. Rose, and Paul H. Gibbs This study assessed the removal of indigenous enteric viruses from raw wastewaters subjected to lagoon treatment. Paired sites lacking mechan- ical aeration (facultative pond systems) were studied in the southeast. southwest and north central regions of the United States. Also included were two systems that provide partial me- chanical aeration of the wastewater in the first pond. Facultative systems were studied during summer, winter and spring, 1979-1980; and aerated systems during the late spring to early fall season, 1979 with sampling performed at all sites concurrently. Intervals between sampling days approximated the mean calculated wastewater residence time for individual cells. Thus, estimates of virus reduction could be made in relation to a common mass of water as well as on a same sampling day basis. Samples were obtained at three points in each system: the raw wastewater and the effluent from both the first and second pond in series. Fecal coliform and chemical/ physical wastewater quality measurements were performed concurrently with virus sampling. The adsorption-elution technique using bentonite clay in the presence of added calcium ion was used to concentrate virus from large (10-30 I) wastewater volumes. Virus samples were reconcentrated in the laboratory, and enteric virus levels were determined on Buffalo green monkey kidney (BGM), He La and human rhabdomyosarcoma (RD) cell lines. In facultative lagoon systems, significant virus reduction occurred throughout the year. Overall virus removal in the first two ponds generally exceeded 99 percent in summer, with combined seasons averages above 95 percent. For all seasons, the first pond of each system provided the greatest contribution to virus removal. There was a statistically significant (P< .05) difference in virus removal on the basis of season, with reduced virus treatability during winter. However, because virus levels in the raw wastewater were lower in winter, the level of virus leaving the lagoons was fairly constant throughout the year. Fecal coliform removals far exceeded virus reductions and removal of other wastewater constituents. Coliforms, total organic carbon (TOC) and sus- pended solids (SS) were, like virus, removed primarily in the first pond of the systems. Statistical analysis did not, however, reveal any wastewater parameters that were highly correlated with virus removal. In partially aerated lagoon systems, the observed percent virus removal during the spring to fall test period was somewhat less than in the facultative systems. However, virus levels leaving both types of lagoon systems were similar. In the aerated systems the greatest removal occurred in second downstream, non-aerated ponds characterized by longer reten- tion times. This Project Summary was developed by EPA's Health Effects Research Laboratory. Research Triangle Park, NC, to announce key findings of the research project that is fully document- ------- ed in a separate report of the same title (see Project Report ordering information at back). Introduction To the authors' knowledge this study is the first quantitative evaluation of the removal of human enteric viruses in oper- ational, full-scale wastewater treatment lagoon systems in which multiple sites, regions and seasons were examined in the United States Work reported by others, using bench, pilot scale or single full-scale lagoon systems has indicated substantial virus reduction The principal thrust of the present study was to deter- mine if this observation is uniform and reproducible, regardless of site, region or season, in typically designed and operated wastewater treatment lagoon systems. It was not the goal of this study to identify specific factors contributing to virus re- moval, though tests were made for statis- tical correlation in other water quality parameters The study consisted of two parts. (1) a larger study encompassing three pairs of wastewater treatment lagoon systems representing widely separated geographical regions within the United States; and (2) a range-finding study, limited to two systems, in which the first one or two ponds in series received partial mechanical aeration The goal of this second study was to determine if there were any differences in overall virus reduction associated with the use of artificial aeration Site selection was preceded by identi- fication of acceptable lagoon design and performance criteria. These criteria were then used in selecting systems with and without mechanical aeration for the various climatic regions. Included in the larger study, using systems without arti- ficial aeration were, two pond systems in the upper midwest, at Beresford, SD, and Lennox, SD; two in the Southeast, at Jonestown, MS and Shelby, MS; and two in the southwest, at El Paso, TX (Northeast Ponds) and Kermit, TX. Study sites using partial mechanical aeration were located at Muskegon, Ml, and Castle Rock, CO In the selection of sites an effort was made to choose systems similar in design, operation, size and wastewater characteristics. Communities without substantial industrial discharge were selected, with the exception of Muskegon, where about 60 percent of the wastewater is of industrial origin, coming largely from paper mills. All systems receive raw, unsettled sewage. The systems serving Muskegon and El Paso serve substan- tially larger populations than the remain- ing systems All systems have at least two ponds operated in series; all have been in service at least 5 years, and all meet EPA discharge standards for biochemical oxygen demand and SS. Calculated combined water retention time for the two ponds in the non-aerated systems varies from 24 days (Kermit) to 1 38 days (Jonestown) with little difference in resi- dence time between the first and second pond in each system and all operate with a continuous effluent discharge. At Muskegon and Castle Rock, residence time in the first or partially aerated portion is much shorter (Muskegon,~2 days, Castle Rock, 6.7 days), while residence in the subsequent non-aerated cells is relatively long (Muskegon,— 150 days; Castle Rock, —63 days) At Muskegon, the large non-aerated lagoons are subject to seasonal drawdown, with the water applied to cropland At Castle Rock, year-round discharge occurs with the final pond in series subject also to additional drawdown for irrigation. The six paired sites without mechanical aeration were studied m each of three seasonal test periods late Julyto October 1979; January to March 1980, and late March to May 1 980. During each season concurrent sampling was performed at three periods m time These sampling times were on approximately 25 day intervals, thereby spanning a period of about 50 days m each season. The two systems using mechanical aeration were studied at only three points in time, representing approximately the begin- ning, midpoint and end of the summer drawdown season Sampling was approx- imately simultaneous at the two sites. On each sampling day, samples for virus concentration and analysis and for physical, chemical and coliform determinations were obtained at three points in each lagoon system. In non- aerated systems these were- (1) the influent wastewater (raw sewage) before entering the first lagoon, (2) the effluent from the first lagoon, or the first pair of lagoons if operated in parallel (Lennox and El Paso); and (3) the effluent from the second lagoon. In systems with mechanical aeration, sampling points were (1) raw sewage; (2) the effluent from the mechanically aerated portion whether one pond (Castle Rock) or two in series (Muskegon); and (3) the effluent from the final pond(s), which were subjected to seasonal drawdown. At the sites with aeration, duplicate samples were taken for virus concentration an< evaluation. The following waterquality parameters other than virus, were determined: SS TOC electrical conductivity, feca coliforms and total dissolved solids (TDS) Also measurements of watei temperature, pH and dissolved oxyger (DO) were made both morning and, when possible, afternoon Preparation of virus concentrate samples in the field as well as measure- ment of fecal coliforms and most physical/chemical water quality parameters were performed by local contractors. For virus concentration m the field the sample volume was 20-35 gal. (76-132 I) for pond effluents and 10-20 gal. (38-761) for raw influent wastewater. Pond effluent samples were simple grab samples.Raw wastewater samples were composites formed by collecting portions of the sample in the early morning, at noon, and the previous evening. Samples were batch-concentrated during the technique of adsorption to bentonite clay in the presence of calcium ion The clay particles were trapped on high solids capacity fiberglass filters, and the loaded filters were shipped under ice in the presence of 2 percent beef extract, pH 9. In the laboratory, virus was eluted from the filters at pH 9 and further concentra- ted by the organic flocculation method at pH 3.5. The floe material was centrifuged, and the centrifuge pellet was then dis- solved with 0.15 M Na2HPO4. Total concentration factors were —4000-fold for pond samples Virus levels in the samples were determined by standard plaque assay procedures using three heteroploid continuous cell lines- Buffalo green monkey kidney (BGM), HeLa, and human rhabdosarcoma (RD). Conclusions and Recommendations At the six paired non-aerated sites, virus levels in influent sewage were highest in summer (mean = 348 plaque- forming units (pfu/l) and lowest in spring (mean = 54.6 pfu/l). This was not reflected in the virus levels leaving the second pond in these systems. These were generally in the range 0.2 to 1.1 pfu/l, though somewhat higher at the Kermit site (1.3 to 5.6 pfu/l) and at the South Dakota sites in winter (2.2 to 10.6 pfu/l). Mean percent removal for the various sites ranged from 99.3 to 99.9 in summer, from 93 9 to 99.1 m winter and from 89.3 to 99.6 m spring. Combined ------- season site means varied from 93.3 at Beresford to 99 6 at Jonestown Results obtained from the various cell lines used were in substantial agreement. Removal of human enteric virus in these systems was consistently good regardless of site, region or season and was comparable to or better than that typically achieved in conventional secondary sewage treatment (without disinfection) Virus removal occurred preponder- antly in the first pond of each system (88 to 98 5 percent as compared to 62 to 69 percent for second ponds) This may be related to the higher levels of particulates available for virus adsorption and settling or to the high levels of bacteria and algae which may have antagonistic interactions with the viruses Similar results occurred for SS, TOC, and fecal coliforms The effect of season, region, site snd season x region on virus removal was studied by analysis of variance (ANOVA) A seasonal difference for the first pond and for the combined ponds were the only effects found significant at the 5 percent level The seasonal difference did not depend on region The better virus removals encountered in summer are in keeping with other work reporting the importance of temperature in virus removal, although in the present study a significant correlation was not obtained. One objective of this study was to measure change in various water quality parameters as the water passed through the lagoons to determine if any were correlated with virus removal No correlations could be detected either within or across seasons Suspended solids and TOC removals were generally good (above 70 percent) only during summer, while fecal coliform removals generally exceeded 99 percent through- out the year Results from the limited study on systems receiving partial aeration in the first ponds indicate no significant differ- ence in the virus content of effluent water between them and the non-aerated systems. Mean effluent values were 0 56 pfu/l for Muskegon and 0 57 for Castle Rock In contrast to the non-aerated systems, virus removal occurred preponderantly in the downstream, non- aerated pond(s). This is attributable tothe very short retention times in the first (partially aerated) portion and the long retention times in the non-aerated portion Virus reduction within the non- aerated (or second stage) ponds at Castle Rock was 97 percent and thus within the range observed for the non-aerated systems. Assessment of percent virus reduction in the Muskegon system was not totally satisfactory because of very low apparent virus levels in the influent sewage. This may have resulted by the presence of materials that interfered with the efficiency of sample concentration or that were toxic to viruses or to the cell monolayers used in their enumeration The presence of 0.2 to 10 pfu/l of enteric virus in pond effluents could represent a low level health hazard if released undismfected to a receiving stream used for recreation or as a drinking water source Disinfection, land wastewater application or the addition of more than two ponds in series with a longer retention time would provide further virus reduction. While the present study provides a general verification of the year-round capacity of wastewater treatment lagoon systems to remove enteric viruses, under typical conditions in the United States, the following should be borne in mind. (1) optimization of this virus removal capability will require a better understanding of the mechanisms involved and their interaction; and (2) the impact of adverse conditions such as excessive loading, extended freezing or toxic chemical inputs has not been evaluated. Studies should be performed to assess the role of virus adsorption to wastewater particulates or to algae and other microorganisms. Other areas which should be addressed in virus removal are the kinetics of virus adsorption, sedimen- tation rates, the effects of thermal turnover and excessive hydraulic loading, the rate of mactivation or irreversible solids association, and the potential for enhanced virus removal by association with settleable solids through addition of clay or cation, reduction in pH or use of coagulant aids. Howart T. Bausum, Stephen A. Schaub. William E. Rose, and Paul H. Gibbs are with the U.S. Army Medical Bioengineering Research and Development Laboratory, Fort Detrick, Frederick, MD 21701. Elmer W. Akin is the EPA Project Officer (see below). The complete report, entitled "Enteric Virus Removal in Wastewater Treatment L a goon Systems," (Order No. PB 83 -234 914; Cost: $10.00, 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 can be contacted at: Health Effects Research Laboratory U.S. Environmental Protection Agency Research Triangle Park. NC 27711 ftUS GOVERNMENT PRINTING OFFICE 1983-659-017/7170 ------- United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 Postage and Fees Paid Environmental Protection Agency EPA 335 Official Business Penalty for Private Use $300 ------- |