United States Environmental Protection Agency Water Engineering Research Laboratory Cincinnati, OH 45268 Research and Development EPA/600/S2-88/024 August 1988 Project Summary of Sludge on Soilborne Plant Pathogens H,A. J, Hoitink and A, F. Schmitthenner Efficacy of composted municipal sludge (CMS) for suppression of Phytophthora root rot of soybean In field soil and for suppression of Rhizoctonta and Pythium diseases of ornamental plants produced In container was investigated over a 3-year period. CMS increased yields of soybean by Improving soil fertility and/or by partial control of Phytophthora root rot {PRR). Disease-enhancing effects of salt in the CMS were controlled with a selective fungicide. In the absence of the fungicide, beneficial effects were obtained only by application of CMS at least 3 months prior to planting with cultivars tolerant to PRR, Increased yields of soybeans were observed when high rates of CMS were applied to corn crops and soybeans were grown in the next year without further CMS application. No residual effects were observed from high rates of CMS applied to soybeans. All container media prepared with CMS cured 4 months or more and stored 4 weeks after formulation became consistently suppressive to Rhizoctonia and Pythium diseases of ornamentals at levels adequate to avoid losses under commercial conditions. An unknown beneficial microflora involved In suppression, if present, survived in the outer low- temperature layer of curing piles only. Several bacterial and fungal isolates were identified that ef- fectively induced suppression to Rhizoctonia and Pythium "damping- off" in CMS media. The relative contributions of these microbes to the overall suppressive effects remain to be determined. Most bacterial strains were more ef- ficacious in combination with an isolate of Trichoderma. A synergistic interaction was found among an isolate of Trichoderma ham a turn and of flavobacterium balustinum for suppression of Rhizoctonia damp- ing-off. These isolates, if added to conducive CMS media, consistently rendered them suppressive in the absence of the 4-week incubation period required for development of natural suppression. This Project Summary was developed by EPA's Water Engineering Research Laboratory, Cincinnati, OH, to announce key findings of the research project that is fully doc- umented in a separate report of the same title (see Project Report order- ing Information at back}. Introduction A significant number of munici- palities in the United States are using the composting process for further treatment of municipal sewage sludge. Composted municipal sludge (CMS) has been utilized effectively as a source of piant nutrients, and the organic matter in CMS may improve physical and biological properties of soils. Severity of a number of plant diseases can be affected by CMS. Some ------- are decreased in severity, whereas others are increased as a result of field soil amendments. The mechanism of this variability is not understood. However, suppression of disease has been associated with increased microbial activity of treated soil. Severa! micro- organisms have been isolated from composts that are involved in the suppression of soilborne diseases in soils or container media amended with composts. Their fate during composting of municipal sludges is unknown. Phytophthora Hoot Rot of Soybeans Phytophthora root rot of soybean is one of the most severe diseases of this crop in the northern soybean growing region. Approximately 10 million acres are infected and can be severely damaged in wet years. Presently, control of root rot is based on resistance of cultivars which has been overcome by new Phytophthora races; application of the fungicide metalaxyt (a potential ground-water pollutant) to soil; or a combination of good drainage, complete soil tillage, rotation, and metalaxyl seed treatment of highly tolerant {least susceptible) cuitivars. The latter inte- grated control package is not available to farmers who wish to reduce soil erosion through conservation tillage, Additional durable and nonpolluting options are needed for control of this important disease. Phytophthora diseases can be controlled by adding certain organic amendments to the sotl. One way to rapidly increase soil organic matter is with manures and CMS. Manure is not readity available to farmers that focus on cash cropping. On the other hand, CMS, derived from municipal sludge, is available in many areas of the United States where soybeans are produced, CMS can be spread on fields with a conventional manure spreader, thus making widespread utilization possible. However, CMS may have a high salt content, which can create adverse effects. Both high fertilizer application rates and high chloride concentrations increase PRR of soybean. The experiments reported here were designed to investigate three areas. First, effects of various CMS rates on root rot severity and yield of soybean in Phytophthora-infested soil were exam- ined. Second, the effect of application timing was determined. Finally, the residua! effects of CMS applied during the previous season were evaluated. Suppression of Rhizoctonia and Pythium D/seases of Ornamenfa/s Produced in Container Media Composts prepared from a variety of tree barks are utilized successfully for suppression of soilborne diseases of ornamental plants produced in container media. These composts are particularly effective for control of Rhizoctonia and Pythium diseases when microbial an- tagonists have recolonized the compost after peak heating has occurred. The mechanisms of suppression involved vary with the pathogen, the source or type of compost used, and the level of decomposition achieved during the composting process. This portion of the study shows that container media amended with CMS can be used ef- fectively for suppression of Thizoctonia and Prrthjum diseases of greenhouse and nursery crops if certain precautions are taken to overcome the detrimental effects imposed on microbial antagonists by the long-term high temperature treatment (>55°C) necessary to ensure fecal pathogen and parasite destruction. In addition, beneficial microorganisms involved in the suppression of Rhizoc- tonia and Pythium diseases were identified, Objectives The objectives of research reported here were: 1} to determine the effects of CMS on soybean PRR severity in the field; 2) to develop procedures for the formulation of container media with CMS that consistently suppress diseases caused by soilborne plant pathogens of ornamental plants; and 3) to determine the nature of the beneficial microflora involved in suppression of these dis- eases, Methods Cftaracferisf/cs of the Compost Composted municipal sludge was obtained from the Columbus Southerly Composting Facility where the aerated static pile composting process has been used on a daily basis since 1980. Raw sludge cake, consisting of a 1:1 ratio (dry wt basis) of primary sludge and waste activated sludge, was dewatered with centrifuges aided by polymer as a coagulant. Sludge cake, averaging 16% to 17% total solids, was mixed with hardwood chips (1 in. to 2 in. chips) at a ratio of 5 volumes of chips to 1 volume of sludge. Mixing was accomplished with front-end loaders in a covered mixing area. The composting process generally was complete after 21 days. Piles were then processed and stacked with or without aeration for a 60-day curing period. Thereafter, the mixture was dried by passive solar drying on a concrete pad. Wood chips were recovered by screening with a 3'8-in. rotary screen Screened compost was then stored in bays in a covered shed- A weekly composited compost analysis was associated with each bay of compost. Compost thus prepared was used throughout this work A summary of the chemical properties of the CMS is presented in Table 1. Soybean Phytophthora Root Rot Field experiments were conducted for 3 years at the Northwest Branch of Ohio Agricultural Research and Development Center (OARDC) of The Ohio State University located at Custar, OH, in a Hoytville clay loam containing 50% clay, 40% si it, and 10% sand The plot area used during the first 2 years was tiled, but gates located at the tile exits were closed to reduce drainage In the third year, the location was changed so that the plots could be irrigated 3 days after planting to provide optimum conditions (or Phytophthora infection This field was tiled, but the tiles were old and nonfunctional Plots consisted of 8 rows, each 26 m long and spaced 76 mm. Each plot was split into two treatments of four rows each. In the first year the split treatments were the cultivars Sloan (some tolerance to PRR) and OX 20-1 (no tolerance to PRR). In the second and third years the split treatments consisted of the cultivar Sloan, with and without 280 g metalaxyl/ha (5.6 kg/ha of Ridomil 5G"). This systemic soil fungicide provides good control of Phytophthora when applied in the seed furrow at planting time. CMS application rates were 0, 10, 20, arid 40 tonne,ha. Application times were preplant, applied within 1 week before planting to maximize salinity effects; and early, applied at least 3 months before planting, either in February or November, to minimize salinity. To determine the fertility effects of trade names or cornrnercidl products does not constitute endorsement of recom- mendation (of use ------- Table ! Characteristics of Municipal Sludge and from the Columbus Southwesterly Compost Sludge" Composted Municipal Sludge Facility Composted Sludge Volatile Solids % Conductivity (mmhoS'Cmff Cl (ng-mi pH W (°.'o, total Kjetdahl) P (°A)t K (%) Ca (%} Mg (%> Cd (fflQtkrj) Cr fmg.kg) Cu (fflQ-kg) Pb (mg-kg) Ni (mg.kg) Zn fmgftgj Na Cmg'ht?; Mean 71 5.8 6.5 3,2 ? 5 8.8 131 218 170 45 1,057 flange 57-79 5.4-6.3 3.2-8,4 1, 6-5.8 0.2-2.2 2,7-26 72-290 1 30-270 92-560 18-74 550-1,900 Mean 59 10,5 370 7.5 4.5 3.3 1.6 3.6 0.86 20 262 255 274 65 1,664 776 Range 43-74 7.5-15,0 245-360 6.2-8.4 3.2-5.3 2.5-4.7 0.9-1.9 3.4-3.8 O.S4-0.8? 73-24 750-325 1 90-640 760-320 54-111 1.000-1,971 699-827 "The sludge was a 1:1 mixture of raw and waste activated sludges. Means are based on 64 samples. Analyses performed by City of Columbus Central Surveillance Laboratory. tDetermined in a saturated pasts extract. tAtt metal concentrations were determined after nitric perchloric ecid digestion. of CMS, plot soil was brought into the laboratory, mixed with the equivalent of 0, 10, 20, and 40 tonne dry wt CMS/ha, planted with soybeans under conditions that promote maximum Phytophthora tosses, and, after 2 weeks, submitted to the Research Extension Analytical Laboratory (REAL), QSU, for fertility analysis {Table 2). Based on this analysis, a fertility control preptant treatment was applied consisting of superphosphate (200 kg PgOs/ha) and NaCI (327 kg/ha) which was the soluble salt equivalent of 20 tonne/ha CMS. Disease analysis consisted of counts of healthy and diseased plants 5 weeks after planting in late October, the weights and percentage moisture of the seed from the two center rows of each pfot were taken to determine effects of Tatjie 2, treatments on yield. Ail treatments were replicated four times. Data were subjected to an analysis of variance with application times and CMS rates as whole plots and cultivars of metalaxyl soil fungicide treatment as split pfots. Preparation of Container Media A CMS container medium was prepared by mixing CMS with Canadian sphagnum peat and perlite (1;2:1,v/v, pH 5.5), This medium was not amended with fertilizer since sludge compost media release adequate amounts of plant nutrients for at least 6 weeks after potting. A peat container medium was prepared from Canadian sphagnum peat and perlite (1:1, v/v) and adjusted to pH 5.5 with a 2;1 (w/w) mixture of cfolomitic and hydrated lime. It was fertilized to adjust fertility to levels identical to that in the CMS medium. The peat medium is conducive to Rhizoctonia and Phthiurn dam ping-off and, therefore, served as a control in this work. Suppression of Pythium and Rhizoctonia damptng-off was deter- mined with cucumber and radish bioassays, respectively. Inoeuta of Rhizoctonia so/an/ and of Pytftium ultimum were added at rates of 0.5 g/L container medium. This amount of inoculum kilted 50% of the plants in a suppresstve medium and all plants in a totally conducive peat medium. Mean disease severity ratings were determined for various sludge compost and peat media to compare efficacy of CMS for suppression of these diseases. Fertility Factors in HoyM/te Stity Clay Affected by Applications of Composted Municipal Sludge Rate (dry tonngfha) 0 10 20 40 LSD (0.05) P 134 214 353 575 93 K 498 501 545 642 55 kg/ha Mn 19 20 28 39 8 Zn 96 105 139 222 58 8 1.8 2.1 2.4 3.1 0,26 WO3 340 370 532 S78 120 ------- Tafile 3. Effect of Composted Municipal Sludge on Yield (kgltia) of Soybeans Under MM Phytophlhora Root Rot Pressure in 1984 Variety Rate (dry tonn&iba) Application Time Sloan (susc) OX 20-8 (very suscj Means Yield (kg/ha) 0 ?0 F0584 2651 2590 7959 230S 1953 2272 20 40 0 10 20 40 Salt Control" LSD =0.05 Feb84 Feb84 May 84 May 84 May 84 May 84 May 84 2849 3345 2779 2657 2923 308? 2583 499 1853 215? 2235 2076 1581 IS58 1612 2356 2751 2507 2366 2252 2322 2087 256 '200 kg of P^Os and 327 kg NaCtha, the soluble salt equivalent of 20 tortna CMS-ha Interactions Among Beneficial Microorganisms in Container Media Bacteria and fungi were isolated by baiting with radish and cucumber roots produced in CMS media or with Rhizoctonia and Pythium soit propagules incubated in sandwiches in the suppressive media. Pure cultures of 650 bacterial isolates and 30 fungal isolates were then tested for their abilities to induce suppression to Rhizoctonia and Pythium damping-off using the bioassays described above. Spontane- ous rifampicin-resistant mutants were isolated from the most effective bacterial strains. The root colonizing potential of these mutants was then tested. Finally. various mixtures of bacterial antagonists with strain 382 of Trichoderma hamatum were examined to reveal synergistic interactions among antagonists in CMS media. Results Soybean PRR In 1984, the 40 tonne dry wt CMS/ha treatment applied in early February significantly increased the yield of the moderately susceptible cultivar Sloan (Table 3), Yield differences were not found in the highly susceptible cultivar, OX 20-8. There were no differences in stand or plant death among any of the variety-treatment combinations. The lack of yield response in the highly susceptible OX 20-8 could be explained by greater hidden root rot damage offsetting a potential fertility response. High rates of CMS applied preplant (May) did not increase yield of the moderately susceptible cultivar Sloan and also significantly reduced the yield of highly susceptible OX 20-8. These results were not accompanied by significant differences in stand or plant death from PRR. The salt-phosphate treatment significantly reduced the yields of both cultivars. This agrees with previous findings for effects of chloride on soybean PRR severity. The salt content of CMS applied preplant probably increased hidden root rot, which prevented a yield response in Sloan and decreased yield in OX 20-8. The yield of Sloan in the high CMS treatment was significantly better than that in the salt-phosphate treatment, indicating a response to organic matter even in the presence of high salts. Cultivar OX 20-8 did not respond to organic matter in the presence of sait. Thus, the response of cultivars to organic matter may have been associated with their level of susceptibility to Phytophthora. In conclusion, the 1984 season demonstrated that the yield response of soybeans may depend on susceptibility to PRR and application time. Possible negative effects of high salt in CMS could be eliminated by early application. To separate the fertility and disease seventy effects, experiments for 1985 and 1986 were changed to incorporate control of Phytophthora with Ridomil, a specific fungicide, Ridomil applied in the furrow with the seed at planting time controls PRR damage in all but the most susceptible cultivars, In 1985 there were no differences between Rrdomil-treated and untreated plots The salt-phosphate treatment had no effect on yield, Thus, in 1985, PRR did not occur Both the 20 tonne ha and 40 tonne ha rates of CMS significantly increased yield (Table 4), The beneficial fertility effects of CMS were clearly evident in this year in the absence of Phytophthora dam age. Residual effects of CMS applied in 1984 were evaluated in 1985. There were no differences among any of the CMS rates in soybean-soybean plots (Table 4), All yield levels were low. In the corn- soybean sequence, the high rale of CMS (40 tonne'ha) applied to corn in 1984 significantly increased the yield of soybean in 1985 Thus, the residual effects of CMS were only evident if applied to corn, which is the logical time to apply CMS in a cropping system. In 1986, only two rates of CMS, 0 and 40 tonne dry wt'ha, were compared. Ridomil, application time, and salt- phosphate treatments also were included. ------- Table 4. Effect of Composted Municipal Sludge on Yield (kg/ha) of Soybean Under Phytophthora-Free Conditions in 19B5 Crop and Time of Application Sludge (tonne/ha) 0 10 20 40 Salt Control' LSD(O.Q5} = 270 Kg Feb 94 Soybean 3073 3086 3060 3154 - May 84 Soybean 2893 2965 2979 3113 2979 May 84 Corn 3370 3423 3423 3558 3370 Nov 84 Soybean 3208 3235 3504 3693 May 85 Soybean 3208 3316 3693 3706 3383 '200 kg of P2O5 and 327 kg NaCUha, the soluble salt equivalent of 20 tonne CMSiha. The crop was irrigated with tO cm water over a 2-day period approximately 3 days after planting when the seedling tap roots were about 5 cm long Flooding induced conditions favoring PRR. Yield levels were not high in this experiment because of a 9-week drought between July and September and fertility benefits were not evident. However, a significant disease response to treatments was evident. Preemergence and postemergence damping-off occurred in plots not treated with Ridomif (Table 5), Stands were poorest in plots with CMS applied preplan! and best in plots treated with CMS early, Ridomit controlled damping- off and resulted in good stands and improved yields in all sludge treatments. In plots not treated with Ridomil, only the early CMS application significantly increased yields compared to the preplant application and the controls, Under this severe disease pressure, early application of CMS increased yields over the control as much as treatment with Ridomil. Response from CMS and Ridomil was not additive, possibly because the late season Table 5. drought prevented a fsrtiiity response. The 1986 data confirm the importance of time of application of CMS. The following conclusions on PRR in soybeans can be drawn from these data. 1. CMS applied just prior to planting may increase Phytophthora damage. Yields will be increased or decreased depending upon severity of Phytophthora. 2. A salt-phosphate mixture equivalent to fertility in CMS, has the same erfect as CMS applied immediately before planting, 3. CMS appfied 3 to 6 months before planting reduced Phytophthora and increased yield. 4. CMS applied in the absence of Phytophthora increases yields even in very susceptible cultivars. 5 Response to 40 tonne dry wt CMS ha is better than to 20 or 10 tonne dry wt ha. 6. Ridomil applied at recommended rates reverses the negative effects of CMS applied immediately before planting. Effect of Compost Curing Time on Suppressiveness of CMS All batches of CMS stored in curing piles with temperatures in the center of the pile higher than 60°C and formulated into a CMS medium, were consistently suppressive to Pythium damping-off. However, these CMS media varied in their effects on Rhizoctonia damping- off- CMS cured 4 months or more in piles and formulated into media, became consistently suppressive to Rhizoetonia damping-off within an additional 4 weeks of storage. The environment in which the compost was cured affected Suppressiveness. CMS stored next to piles of bark compost suppressive to Rhizoctonia damping-off, became sup- pressive earlier than CMS stored in isolated locations. Microorganisms iso- lated from the suppressive CMS that could cause this effect were Trichoderma spp. and a number of bacterial antagonists. Without this recolonization of CMS with antagonists of R. solani, Suppressiveness did not develop. Effect of Composted Municipal Sludge on Stands (ptants.26 m of Row Spaced 76 cm) and Yields (kgtha) of Sloan Soybean Under Severs Phytophthora Root Hot Pressure in 1986 Stand (plants 26 m rows) Yield (kg ha) Sludge (tonne; ha) 0 40 0 40 Salt Control" Time of Application Feb 86 Feb 86 May 86 May 86 May 86 No Rid omit 211 226 176 105 207 Ridomil 352 318 346 312 354 Mean 281 272 261 209 280 No Ridomil 984 2056 627 512 815 Ridomil 2420 2615 2251 2736 2440 Mean 1705 2332 1442 1624 1624 LSD (0.05) 89 607 337 "200 kg of P2&5 and 327 kg NaCl^ha, the soluble salt equivalent of 20 tonne CMS'ha ------- Table 6 Suppress we ness of a Nursery Sludge Compost and a Conducive Peat Container Medium to Pythium and Thizoctoma Damping-Off Over a 2-year Period Disease Seventy Rating? Medium Sludge Compost Peat Control Treatment Chech Infested Check infested May 7.7 1.8 1.2 3.9 July 1.0 2.6 1.1 4.0 Nov 1.0 23 17 3.5 LSD (P =0.05) Oct 1 2. 1 3. 0.8 7 7 2 a May 1.5 3.7 r.3 3.9 July 1 2 7. 3 2 6 3 9 0.3 Nov 12 23 1 7 36 Oct 1 r 29 ! 1 38 "Infested with 0.5 g Rhizoctonia solan! or Pythium ulttmum soil inoculum per L container medium. fOisease seventy ratings: 1 = symptomless; 2 ^diseased but not damped-off. 3 =postemergence damp/rrg-otf; 4 =pre emergence damping-off, based on five pots each planted with 32 radish seed. May, July and November represent 1984. October represents 1985 values. Suppression on Diseases During the Production of Potted Crops Pythium diseases caused by P. ultimum or P. aphanidermatum in floricultural pot crops were suppressed in CMS media throughout a 5-month production period. Similarly, Pythium root rot caused by P. irregulare in nursery crops was suppressed over a 2-year production period (Table 6), Diseases caused by R. solani were also suppressed in both floricultural (Figure 1} and nursery crops (Table 6), but only consistently if the formulated container medium was stored 4 weeks before utilization. It was concluded that CMS becomes naturally suppressive to Pythium damp- ing-off but that 4 months of curing is required followed by an additional 4- week storage period of the formulated medium required to ensure suppres- siveness to Rhizoctonia dampmg-off Efficacy of Microbial Antagonists T. hamatum 382 added as an antagonist by ttself to a conducive CMS medium did not induce a significant level of suppression to Rhizoctoma dampmg- off (Table 7). However, a combination of Table 7. Suppression of Rhizoctonia Damping-Off Induced by Bacterial Antagonists Alone and in Combination with Tnchoderma hamatum in a Sludge Compost Container Medium Disease Severity Patmgt Bacterial Antagonist" LSD 005 Bacterial Antagonist Alone Bacterial Antagonist with T, hamatum Wone Bacillus cereus 106 Enterobacter cloacae 127 0. cloacae 3)3 Flavobacterium balustinum 299 (ATCC53198) Janthinobactenum livid urn 275 Pseudomonas ftuorescens biovar til, A9i P. (lucrescens biovar V, Ai P. fluorescons biovar V, A498 P. putida 305 P. putida 375 P. putida 37J P. stut2ert280 Xanthomonas maltophilia 76 (ATC53199) 2.9 3.0 2.6 21 3.1 2.7 2.9 2.4 2.7 22 23 23 24 30 26 20 2.6 2 J 2.1 25 2.4 26 23 1 3 2 1 25 27 2.4 04 "Bacterial antagonists were adcfed to the heated (60"C, 5 days) sludge compost container medium at initial population levels of 10SCFU g dry wt T. hamatum was added af TO4 CFUg dry wt. flnfested with 0.5 g Rriizoctonia solatu soil inoculum:L container medium. Mean disease seventy rating determined 7 days after incubation at 26"C from Five pots planted with 32 radish seeds each. 1 -symptomless^ 2 -diseased but not damped-off: 3 -postemergence damping-off; and 4 -preemergence damping-off Mean disease severity in the container medium not infested with R. solani was 1.2 ------- 8.O- Aug Sept Oct Nov Dec Sampling Date Figure 1. Suppresstveness of s peat and a sludge compost medium {CMS} to Rhizoctonia damping- off during production of poin- settta plants. Disease severity rating: 1 ~symptomtess; 2-dis- eased but not damped-ofl, 3=post-emergence damping- off; and 4-^preemergence damp- ing-olf. Infested with 0.5 g Rtzoctonia inoculum per liter container medium. ISOoos for all sampling times com- b/t-ied-0.4. Flavobacterium balustinum strain 299 with T. hamatum 382 significantly (P = 005) reduced damping-off, even though each bacterial antagonist by ttseif was not effective. This bacterial antag- onist significantly enhanced population development of the T. hamatum strain early after its addition to the compost- amended medium (Figure 2). A syner- gistic effect was established, therefore. This effect occurred in compost cured 4 months that was conducive if not infested with the antagonists Several other combinations of bacterial antagonists and T. hamatum 382 were identified that had similar synergistic interactions in sup- pression of Pythium damping-off in the CMS medium. It was concluded that microbial antagonists can be utilized to develop predictable levels of suppression to Rhizoctonta damping-off in container media amended with conducive CMS. This process eliminates the need for storage of formulated CMS media. Tho full report was submitted in fulfillment of Cooperative Agreement No, CR-810581 by the Ohio State University under the sponsorship of the U.S. Environmental Protection Agency. 4.0 z. 0 ? 8 15 Days After Inoculation with Antagonists Figure 2. Population development of Fia- vobactenum balustinum 299ft f— ) and T. hamstum 382 ( I in a container medium planted with cucumber and amended with compost F. baiustinum and T. hamatum were inoculated individually in a container medium amended with (1) conducive compost from the high temperature center of the curing pile { 01; (2) svppres- stve edge compost { B I; of 13) were introduced as a combina- tion (reaiment in the conducive center compost medium ( © ) Vertical bars indicate standard error. ------- H.AJ. Hoitink, and A.F Schmrtthenner are with the Ohio State University, Ohio Agricultural Research and Development Center, Woosier, OH 44891 James A. Ryan is the EPA Project Officer (see below). The complete report, entitled "Effects of Composted Municipal Sludge on Soiihorne Plant Pathogens," (Order No. PB 88 195 714-AS; Cost $14.95, subject to cfhange) will be avtitabte only from: National Technical Information Service 5?85 Port Hoya/ Road Spring field, VA22161 Telephone: 703-4S7-4SSO 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 Official Penalty for Private Use $300 EPA/6QO/S2-88/024 ------- |