United States Environmental Protection Agency Water Engineering Research Laboratory Cincinnati OH 45268 Research and Development EPA/600/S2-87/028 June 1987 Project Summary Survival and Transport of Pathogens in Sludge-Amended Soil: A Critical Literature Review Charles A. Sorber and Barbara E. Moore A study was undertaken to critically review available information on the survival and transport of pathogens from municipal wastewater sludges ap- plied to land. Unfortunately, the amount of quantitative, comparable data related to pathogen behavior in sludge-amended soils is extremely lim- ited. Most available data are restricted to Salmonella and indicator bacteria. In general. Salmonella showed a 90% (T90) reduction within 3 weeks in sludge-amended soils. In warm cli- mates, inactivation of viruses near the surface was quite rapid, with a median Tgo of 3 days. However, at low tempera- tures, T90 values of approximately 30 days were observed for viruses. Maxi- mal parasite survival, as determined by Ascaris ova recovery, was relatively long near the surface, with a median Tgo of 77 days. Extremely limited vertical movement of some pathogens may be anticipated in sludge-amended soils. Although monitoring at sludge application sites has not revealed that sludge amend- ment affects the bacterial quality of groundwater, limited transport of indi- cator bacteria to depths up to 180 cm has been reported. Under field condi- tions including exposure to natural rainfall, virtually no viruses have been detected in soil-water percolates. Avail- able literature strongly favors the con- tention that parasitic ova are retained at the point sludge is introduced to the soil. Finally, insufficient data are avail- able for adequate modeling of patho- gen survival and transport in sludge- amended soils. This Project Summary was devel- oped by EPA's Water Engineering Re- search 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 An integral part of almost any waste- water treatment plant is the sludge management system. Residual solids are produced in nearly every unit proc- ess associated with conventional waste- water treatment. Approximately 6.5 mil- lion dry tons of municipal wastewater sludges are generated annually in the United States. By the year 2000, the quantity of municipal wastewater sludge produced is projected to almost double. Sludge management currently accounts for approximately half of the cost of wastewater treatment. Thus a major goal is to reduce the costs of sludge handling. Equally important is to reduce to an acceptable level the risks to public health, safety, and welfare that arise from and are otherwise associated with sludge disposal. Among several disposal alternatives, land application of sludge is increasing in popularity. Indeed, there is every rea- son to believe that the practice will expand at a greater rate in the years to come. The presence of infectious mi- ------- croorganisms in sludges may, however, place certain constraints on their use on land. The concentration of pathogens in wastewater and thus in wastewater sludges is influenced by a number of factors, including the age and health of the contributing population, population density, sanitary habits, and the season of the year. Microorganisms of public health concern are generally classified into three broad categories: bacteria, viruses, and parasites. Parasites are often further differentiated into helminths and protozoans. Hundreds of organisms fall into these categories and may be present in domestic waste- waters. A wide variety of disease-causing mi- croorganisms known to be transmitted by the fecal-oral route may potentially be transmitted through environmental exposure. A more focused list of micro- bial agents can be prepared, however, with the application of additional crite- ria such as demonstrable presence in wastewaters or sludges and/or docu- mented environmental transmission of disease. Table 1 provides such a listing. Although some of the organisms listed are overt pathogens, reports of their oc- currence in wastewater and sludges in the United States are quite rare. How- ever, advances in microbiological and medical sciences may identify addi- tional pathogenic organisms linked to environmental disease transmission. Wastewater treatment affects the var- ious organism types in different ways. In general, microbial segregation oc- curs during conventional wastewater treatment. Bacteria, viruses, and some parasitic cysts tend to become associ- ated with the sludge component, as do the heavier eggs of certain parasites such as Ascaris. Conventional sludge treatment processes can reduce the lev- els of sludge-associated pathogens. In the absence of extensive treatment, however, wastewater sludges will con- tain measurable concentrations of these microorganisms. Thus from a public health standpoint, applying wastewater sludge to land needs regulation in re- gard to the pathogens known to be present in these sludges. Interim regulations relating to sludge treatment and disposal have been de- veloped and were published in 40 CFR Part 257. These current regulations are based on the expected operational per- formance of specific unit processes and on the absence of health effects directly related to land application practices. Ul- timately, however, regulations should be founded on both a complete under- standing of the fate and transport of pathogens in sludge-amended soils and on the epidemiological implications as- sociated with the numbers of organ- isms to which humans are subjected as a result of these practices. To facilitate the development of scientifically based regulations, a critical review was made of available information on the survival and movement of pathogens from mu- nicipal wastewater sludges applied to land. Methods Acquisition of Literature Extensive literature searches were conducted, and a significant number of documents were accumulated from a variety of sources. The search for rele- vant documents was carried out in four steps: primary (database) searches, secondary searches, author contacts, and manual searches. A total of 12 primary literature data- bases were searched. This step resulted in the acquisition of a total of 819 titles and abstracts. After they were read and duplication was eliminated, 95 unique documents were identified for hard- copy acquisition. The secondary literature searches were begun when the hard copies of documents from the primary searches were available for review. All references in the primary documents were consid- ered possible secondary sources. Fur- thermore, each document from this sec- ondary search represented possibilities for the identification of additional litera- ture sources. Contacts were made with authors identified as major contributors to the literature obtained during the primary and secondary searches. In addition, personal contacts were made at a num- ber of national meetings. These efforts proved fruitful, as they obtained a num- ber of obscure and unpublished docu- ments of value to the study. Manual searches were made of se- lected current science and engineering journal issues. The journals selected for manual searches were those that yielded documents relevant to the study through the primary and secondary lit- erature searches. Guidelines for Literature Evaluation The literature review encountered a broad spectrum of studies ranging from investigations employing exogenously added organisms to monitoring of in- digenous organisms at field sites. The Table 1. Organisms of Major Concern in Land Application of Municipal Wastewater and Sludges Group Name of Organism Primary Disease Remarks Bacteria Viruses Helminths Protozoans Legionella pneumophila Salmonella sp. Shigella sp. Vibrio cholerae Hepatitis A virus Non-A, Non-B hepatitis Norwalk-like agents Rotavirus Ascaris sp. Giardia lamblia Acute respiratory disease Gastroenteritis, typhoid and paratyphoid fever Bacillary dysentery Cholera Infectious hepatitis Hepatitis Gastroenteritis Gastroenteritis Ascariasis Giardiasis (Gastroenteritis) Aerosol transmission documented, but no cases linked to wastewater exposure to date Overt pathogens but low probability of occurrence in wastewater in the United States Documented waterborne transmission Preliminary evidence for waterborne transmission Documented waterborne transmission Documented waterborne transmission Documented waterborne transmission ------- challenge posed by such diverse experi- mental conditions was to qualify the data within a common framework, re- flecting (insofar as possible) expected responses in natural systems. To en- sure a relatively unbiased appraisal of existing literature, guidelines were set to critically evaluate both laboratory and field studies before beginning the review of individual reports. For example, attention was focused on the appropriateness of both sam- pling and analytical procedures for the recovery and identification of specific organisms. The adequacy of the experi- mental design was evaluated with re- gard to the number and frequency of samples collected as well as the control procedures used. From a regulatory and design standpoint, the collection of sup- porting data during the course of exper- imentation or monitoring could provide valuable information; thus particular at- tention was given to ancillary data that might affect pathogen survival or trans- port or both. Specifically, the collection of environmental data in the areas of temperature, rainfall, and various soil parameters was deemed important. Finally, to provide a common frame- work within which organism survival could be addressed, simple inactivation values representing 90% (T90) and 99% (T99) dieaway were graphically deter- mined. For this purpose, minimal crite- ria were established for using published data: initial monitoring of amended soil must have occurred within 2 weeks of sludge application, and a minimum of three positive, quantitative recoveries must have been recorded over a con- secutive monitoring period. In addition, there must have been no data extrapo- lation beyond the actual sampling pe- riod. Results and Discussion In considering the various studies de- tailing the survival and transport of mi- croorganisms in sludge-amended soils, the limitations influencing quantitative results in such systems must be recog- nized. Perhaps the most important problem in evaluating the behavior of microbes in sludges and soils is the methods used for organism recovery. Though standard methods for detecting indicator bacteria in water and waste- water have been widely applied in soil systems, these bacterial groups are not normally associated with human dis- ease. The recovery and enumeration of bacterial pathogens, viruses, and para- sites often require elaborate procedures involving a high degree of technical competence and experience. In addi- tion, the factors affecting organism re- covery from sludge and soil systems are not well understood. Furthermore, the recovery of viable bacteria, viruses, and parasites is limited by the volume of sample that can be analyzed, thus im- posing, in some instances, a restrictive sensitivity limit on organism detection. Survival of Microorganisms in Sludge-Amended Soils Factors frequently cited as affecting microbial survival in soil include soil moisture content, temperature, pH, sun- light, organic matter, and antagonistic soil microflora. Microbial pathogens in- troduced into the soil by sludge amend- ment will be influenced by these factors. However, the nature of the sludge- amended soil environment may moder- ate soil conditions in ways that could affect the survival of microorganisms. For example, sludge application may dramatically increase the organic con- tent, nutrient content, and moisture- retention capability of sandy soils. In ad- dition, soil pH could be influenced by added sludge or management practices such as lim>ng. Even soil temperature could be affected by the surface applica- tion of sludge. Though the interplay of these factors in sludge-amended soil may favor organism survival in some cases, more rapid pathogen inactivation may occur in other situations. As shown in Table 2, T90 values for Salmonella survival in sludge-amended soil fall within the range of 3 to 61 days, with median values of 12 and 8 days for soil depths of 0 to 5 cm and 5 to 15 cm, respectively. A closer review of selected studies reveals a seasonal trend of bac- terial inactivation. When salmonellae in sludge-amended soils were subjected to winter conditions, T90 values of 12 to 15, 17, and 22 to 61 days have been esti- mated in three published field studies. Similarly, at a temperature of 12°C, Salmonella sp. were observed to decay with a T90 of 8 to 11 days in a controlled laboratory study. During summer expo- sure, a much more rapid inactivation of indigenous salmonellae has been ob- served with T90 values of 4 and 6 days in two separate field studies. Experiments conducted during warm growing sea- sons with laboratory-grown strains of Salmonella have resulted in T90 values of 6 and 10 days in an Ohio study and 14 days in a Norway study. Hence studies with both indigenous and seeded salmonellae are in relatively good agreement, showing (with one excep- tion) a 90% bacterial reduction within 3 weeks of sludge application. The exceptionally long survival times often cited for Salmonella persistence actually arise from seeded studies in which high levels of bacteria ranging from 106 to 1010/L were added to sludge before land application. Under these conditions, and assuming a maximum T99 of 45 days, persistence times in ex- cess of 5 months could be anticipated. If growth or regrowth of seeded orga- nisms occurs, this survival time could be substantially longer. On the other hand, indigenous salmonellae at actual field sites have generally persisted at low levels for less than 2 months, al- though a few positive recoveries have been reported as long as 3 to 5 months after sludge application. Most published literature documents the behavior of indicator bacteria in sludge-amended soils. With the excep- tion of one study, 90% of the fecal coli- forms could not be recovered within 6 weeks of sludge application. A 90% loss of fecal streptococci occurred with 4 weeks. Although the number of studies is more limited, total coliform bacteria displayed significantly slower inactiva- tion rates, with T90 values generally twice those of the other bacterial indica- tor groups (Table 2). Note, however, that T90 values for total coliforms are polarized, with most values ranging from 14 to 42 days and a second group ranging from 129 to 172 days. An evalu- ation of the overall survival results of these groups of bacteria reveals a di- chotomy with the seasonal survival of coliform bacteria as reported by two re- search groups working in the Pacific Northwest region of the United States. Although the actual Tgo values were dra- matically different, both studies ob- served longer survival times during warmer months than during cooler months. Coliform regrowth is the most likely explanation of these findings. In- deed, regrowth of coliform bacteria in the spring following a decrease in levels during the winter has been reported. These results highlight the difficulty in evaluating bacterial inactivation when organisms are capable of replication. In- terpretation of data for indicator bacte- ria is further complicated by the fact that several bacterial species, including unique soil microflora, may be recov- ered by the analytical procedures used. These bacterial populations do not nec- essarily share the same inactivation or regrowth characteristics. ------- Table 2. Summary of Microorganism Survival in Sludge Applied to Soil Die-off—T90 (days) Die-off—T99 (days) Bacteria Salmonella Fecal streptococci Fecal coliforms Total coliforms Viruses Parasites Depth (cm) 0- 5 5-15 0- 5 5-15 0- 5 5-15 0- 5 5-15 0- 5 5-15 0- 5 5-15 Minimum 6 3 7 72 7 4 16 9 <1 12 17 Maximum 61 22 28 30 84 49 172 70 30 56 270 Data unavailable Median 12 8 14 20 25 13 85 17 3 30 77 Observations 10 17 9 11 19 12 6 12 9 4 11 Minimum 11 7 14 30 12 9 28 18 2 60 68 Maximum 45 45 63 60 165 90 350 40 52 100 500 Median 22 18 24 40 53 32 155 32 6 60 81 Observations 8 15 8 10 16 11 4 9 6 3 5 Data unavailable Survival of viruses in soils is influ- enced by many of the same parameters described for bacteria. The effect of temperature on the survival of viruses is well documented: lower temperatures favor longer survival. Furthermore, an optimal soil moisture content favors virus survival in soil, whereas desicca- tion results in a more rapid loss of viruses. Also remember that viruses (as obligate intracellular parasites) do not replicate outside of an appropriate host. Thus data characterizing their survival in sludge-amended soil are perhaps more straightforward. Viral T90 values have ranged from less than 1 day under hot summer condi- tions to 56 days in the winter. Note that data presented in Table 2 that appear to support extended viral survival with soil depth actually reflect sampling to 20 cm in one Danish study where an average temperature of 0.5°C was observed. Un- doutstedly, viruses can persist for ex- tended periods in the soil environment where cold temperatures favor their survival. Studies completed under win- ter conditions in both Denmark and Ohio showed very similar inactivation rates, with T90 values of 30 days for two different enteric viruses. As evidenced by available data, however, inactivation at the soil surface can be quite rapid when viruses are exposed to high tem- peratures and drying conditions such as those prevailing in the southern United States during the summer and fall. Among the parasites, protozoa seem to be very sensitive to drying, and under these conditions, survival rates are usu- ally short. However, ova of helminths such as Ascaris are quite resistant to en- vironmental stress. Parasites are also unable to replicate outside of their ap- propriate animal or human hosts. Only three independent research groups have reported quantitative data for parasite survival in sludge-amended soil that allows estimation of inactiva- tion rates. All but one Tgo value is based on the addition of exogenus Ascaris or Toxocara ova to the sludge-soil system. As expected, T90 values for these para- sitic forms exceeded those of all other microbial groups, ranging from 17 to 270 days, with a median value of 77 days (Table 2). Seasonal effects on ova survival were observed. Following sum- mer sludge application, Ascaris ova were inactivated with apparent T90 val- ues of 17 days in one study. Survival after applying sludge in the fall at the same location was more extended, with a T90 value of 65 days for Ascaris ova and 77 days for Toxocara ova. From an- other report, a T90 value of 30 days was estimated for Ascaris in sludge sprayed onto an untilled plot in the summer, whereas 90% inactivation following winter application required 80 and 90 days in separate experimental plots. Notably, after winter sludge application in this study, Ascaris ova survived dra- matically longer, with a T90 of 200 days in tilled plots planted with a cover crop in the spring. Presumably, this ex- tended survival time was favored by de- creased soil temperature resulting from crop shading and/or by higher soil moisture resulting from irrigation and rainfall. Attempts were made to analyze statistically the available quantitative data. Unfortunately, sufficient data were available only for temperature and die-off. Least-squares regression analy- ses of raw and transformed data were performed for the die-off recorded for salmonellae, fecal streptococci, fecal coliforms, total coliforms, viruses, and parasites. Only for fecal coliforms were there sufficient data to discriminate be- tween soil depths. In the cases of salmonellae, viruses, and parasites, die- off at all depths was used in the analy- sis. Die-off at 5 to 15 cm was used in the analysis of salmonellae, fecal strepto- cocci, and total coliforms. Poor correlation was observed be- tween organism inactivation and tem- perature for salmonellae, fecal strepto- cocci, total coliforms, and parasites, whereas very good correlation was ob- served for fecal coliforms at both depths and for viruses. Results of this analysis for viruses appear in Figure 1 and illus- trate some of the limitations of the avail- able data. More often than not, the transformed T90/T99 data correlated bet- ter with temperature, but the difference was not judged to be significant. Close scrutiny of these data shows that usable information for microorganisms such as viruses was available only at temper- ature extremes. Or, in other words, data tended to be clumped at either the warm or cold ranges, with few data in between. This observation restricts the value of an analysis over a range of tem- peratures and suggests the need for more detailed evaluation. One approach to this evaluation was to use nonparametric correlations that make no assumptions about the nor- mality of the distribution of the vari- ables. The Kendall rank-order correla- tion was chosen for this evaluation. Only fecal coliform data at both the 0- to 5-cm depth and 5- to 15-cm depth were judged to be significant at the 5% level. Transport of Microorganisms from Sludge-Amended Soils In addition to survival of pathogens in sludge-amended soils, consideration must be given to their ability to move in this environment, either into surface waters through runoff or, perhaps more important, into groundwater through the soil profile. Though runoff may be viewed as largely the physical transport ------- Correlation Coefficient = 0.906 1 . Correlation Coefficient = 0.542 10 20 Temp., °C 10 20 Temp., °C 30 Correlation Coefficient -0.913 Correlation Coefficient = 0.987 10 20 Temp., °C 10 20 Temp., °C Figure 1. Least squares regression plots for temperature and virus survival at all depths. of microorganisms associated with par- ticulate material, vertical microbial transport is more complex. Exceedingly few studies have ad- dressed the presence of microorga- nisms in runoff from sludge-amended soils. No significant bacterial or viral im- pact has been observed on surface water at actual sludge application sites. However, as long as viable bacteria were present in sludge-amended soil, they were recovered at elevated levels from runoff intercepted at the lower end of sludge-amended test fields. Simi- larly, parasitic ova have been recovered from irrigation return flow at a sludge application site. Obviously, sludge ap- plication methods that minimize the dis- placement of sludge in surface runoff should be used if microorganism trans- port in runoff is to be avoided. Removal of bacteria from wastewater percolating through a soil is due to both mechanical action (i.e., straining or sieving at the soil surface) and adsorp- tion to soil particles. Similarly, the phe- nomenon of adsorption as a mecha- nism for retaining viruses in soil systems has been demonstrated. Re- lease and movement of these microor- ganisms would be expected, since physical adsorption to particulates is a reversible phenomenon and, in part, ion-dependent. The transport of protozoa and helminths in soils appears to be more limited than for bacteria or viruses. This may be the result of the considerable size differences between viruses, bacte- ria, and parasites. For example, proto- zoa are up to 20 times larger than bacte- ria and up to 2,000 times larger than enteroviruses. Ascaris eggs are even larger. Clearly, mechanical straining may be the most important factor gov- erning the transport of these parasites. Relatively few studies conducted at sludge application sites have looked for vertical transport of microorganisms. Limited monitoring has shown no demonstrable impact of sludge applica- tion on fecal coliform levels in ground- water. However, coliform bacteria have been detected sporadically at shallow depths of 100 and 180 cm beneath sludge-amended sites in the northwest- ern United States. In contrast, viruses have been recovered from relatively deep wells (8.5 and 18 m) at one sludge disposal site in Florida, whereas the re- sults of groundwater monitoring at a second location in Florida were nega- tive for viruses. Comparison of such re- sults at operational field sites is often impeded, however, by the fragmentary nature of available data. Few studies have conducted integrated, long-term monitoring for viruses in which sludge, sludge-amended soil, and groundwater sampling were coordinated. Available literature strongly favors the contention that parasitic ova are retained at the point of sludge introduction. Ground- water monitoring for parasites has not been conducted and seems unneces- sary given the relative size of most para- sitic forms and their observed retention in the upper soil profiles. Notwithstanding these limited field data, laboratory studies have demon- strated some transport of bacteria and viruses through sludge-amended soils. A single study has demonstrated move- ment of Yersinia, fecal coliforms, and fecal streptococci through 46 cm of soil, although maximal levels represented less than 0.1% of the bacteria present in the sludge. Note that experimental con- ditions in this study were chosen to rep- resent a worst-case situation in which a total of 46 cm of rainfall was applied over a 3-week period. Several laboratories have studied the vertical movement of viruses in model soil columns or cores. Studies in which lysimeters or cores amended with virally contaminated sludge have been exposed to natural rainfall have con- firmed results obtained by groundwater monitoring at most sludge application sites. Specifically, in two separate stud- ies, very few viruses were detected in soil water percolates intercepted at depths of 54 and 125 cm. Under certain laboratory test conditions, however, viruses applied with sludges have been transported through soil depths ranging from 13 cm in one study to 46 cm in a second study. When compared with the movement of free viruses, sludge- bound virions are much more effec- tively retained, apparently within the sludge-soil matrix at the point of appli- cation. Conclusions 1. The number of quantitative, com- parable data describing pathogen survival or transport in sludge- amended soils are extremely small. Survival data are available only for Salmonella sp., selected enteric viruses, and Ascaris ova, and studies on pathogen transport are limited to Yersinia sp. and certain viruses. 2. Where adequate quantitative data exist, these observations can be made: • Inactivation of indicator bacteria as described by median T90 values was greater than that observed for Salmonella. • Viral inactivation appears to be faster than Salmonella inactivation near the soil surface. However, all but one study used to estimate viral die-off were conducted in rather narrow temperature ranges, thus ------- highlighting a potential bias in the application of these values. • Inactivation of parasites near the soil surface is relatively slow, per- haps as much as 5 times slower than Salmonella inactivation and more than 13 times slower than virus inactivation. 3. Exceedingly long survival times somtimes cited for Salmonella arise from studies in which high levels of added organisms (106 - 1010/L) were present. 4. The only strong evidence for bacte- rial regrowth in sludge-amended soils is related to organisms of the coliform indicator group. 5. Of the physical and meteorological parameters considered, only temper- ature could be correlated with mi- croorganism survival. 6. Inadequate data exist to assess criti- cally the vertical transport of patho- gens from sludge-amended soils. However, several general observa- tions can be made: • Data collected at all but one opera- tional field site have not demon- strated a deterioration of ground- water quality related to sludge application. • Selected studies have documented limited bacterial movement to depths of 180 cm beneath sludge- amended soil. • Limited laboratory studies suggest that viral retention is enhanced in sludge-amended soils compared with effluent-irrigated soils. • The size of parasites appears to pre- clude their vertical movement from sludge-amended soils, but studies designed to address this question were not found. 7. Exceedingly few studies have ad- dressed the issue of microorganisms in runoff from sludge-amended soils. However, there is a high probability that uncontrolled runoff wil contain pathogens as long as viable orga- nisms are present in sludge- amended soils. 8. Insufficient data are available for ad- equate modelling of pathogen sur- vival or transport in sludge-amended soils. Not only are microbial results limited, but prevailing environmen- tal and soil conditions have not been adequately documented in many published reports. Recommendations The following specific recommenda- tions are designed to obtain the data required to formulate a more complete understanding of the survival and/or transport of pathogens in sludge- amended soils: 1. Studies specifically designed to develop such comprehensive data should be conducted. 2. These studies should be restricted to representative pathogens such as Salmonella, selected human enteric viruses, and parasites in- digenous to municipal wastewater sludges. 3. Though it would be desirable to conduct such studies under field conditions with indigenous orga- nisms, this approach may be lim- ited by the levels of pathogens in sludges coupled with the relative insensitivity of currently used de- tection methods and the existence of a wide variety of uncontrolled environmental variables. 4. The use of selected seeded orga- nisms in sludges under closely controlled laboratory conditions may be the most reasonable ap- proach. 5. Laboratory experimentation must be carefully designed to simulate a range of temperature, moisture, sludge loading conditions, and soil types found nationwide. The full report was submitted in fulfill- ment of Cooperative Agreement No. CR811918-01-0 by The University of Texas under the sponsorship of the U.S. Environmental Protection Agency. Charles A. Sorber and Barbara E. Moore are with the University of Texas at Austin, Austin. TX78712. Albert D. Venosa is the EPA Project Officer (see below). The complete report entitled "Survival and Transport of Pathogens in Sludge- Amended Soil: A Critical Literature Review." (Order No. PB 87-180 337/ AS; Cost: $18.95, 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: Water Engineering Research Laboratory U.S. Environmental Protection Agency Cincinnati, OH 45268 ------- ------- United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 BULK RATE POSTAGE & FEES PAID EPA PERMIT No. G-35 Official Business Penalty for Private Use $300 EPA/600/S2-87/028 ------- |