NEW YORK STATE WATER RESOURCES INSTITUTE Department of Biological and Environmental Engineering

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1 NEW YORK STATE WATER RESOURCES INSTITUTE Department of Biological and Environmental Engineering 230 Riley-Robb Hall, Cornell University Tel: (607) Ithaca, NY Fax: (607) Methane and Nutrient cycling in Septic Leach Field Systems Cristina Fernandez-Baca, Civil and Environmental Engineering, Abstract Onsite septic systems treat approximately 25% of U.S. domestic wastewater. Despite their prevalence and continued use in new building, few studies have attempted to characterize septic systems air and water quality impacts. Understanding septic systems effectiveness is vital to managing them in a way that promotes both air and water quality. Systems that are improperly sited and/or managed can cause ground and surface water contamination as well as increased greenhouse gas (GHG) emissions as compared to well-managed systems. To examine microbial populations and potential GHG and nutrient cycling within leach field soil systems, we constructed two leach field soil columns in the lab. Reactors were subjected to either flooded conditions (Column A) or well-maintained conditions (Column B) and compared in: (1) measured atmospheric methane (CH 4 ) fluxes; (2) measured CH 4 depth profile, (3) distribution and activity of key organisms involved in CH 4 cycling; (4) measured chemical oxygen demand (COD) and nutrient treatment (N, P). Overall, the columns performed more similarly in nutrient removal than in CH 4 cycling with flooded conditions significantly increasing CH 4 fluxes and overall CH 4 production. COD removal was variable and is negatively impacted by flooding while nutrient removal appears to be unaffected by flooded conditions. Three Summary Points of Interest Methane emissions from flooded septic leach field soils are significantly higher when compared to wellmaintained systems. Overall, flooded systems create more methane that is not consumed by microbial communities. Nutrient (nitrogen and phosphorus) removal is not affected by flooding, however COD removal is variable and does appear to be negatively affected by long-term flooding. Abundance of microbial populations involved in methane cycling were affected by flooding. With methane producing organisms more abundant in flooded conditions. Keywords Greenhouse gases, septic systems, biomarkers, COD (chemical oxygen demand)

2 Introduction On-site septic systems currently treat wastewater from approximately 25% of U.S. households (US EPA 2013). Septic systems are a practical and inexpensive means of decentralized wastewater treatment for rural and periurban areas. They are able to achieve similar treatment levels as their larger, centralized, and more energyintensive wastewater treatment plant counterparts while still maintaining many of the same public health and environmental benefits (US EPA 2012; US EPA 2014). Despite the popularity and numerous advantages of these decentralized systems, there is a fundamental lack of information on how they effect the surrounding environment. In particular, their impact on water quality and potential greenhouse gas (GHG) emissions is poorly understood. Septic systems are popular in part due to their low operating and maintenance requirements. However, because these systems require such infrequent service and are inconspicuous by nature, failures can arise and remain unnoticed and thus un-repaired. In particular, failed septic systems causing ground and/or surface water contamination are difficult to identify and diagnose. Consequently the number of failing septic systems in the U.S. is unknown. Complicating the issue is the fact that there is no strict definition as to what constitutes a failing system (US EPA 2002). Currently there are no requirements for states to collect data on septic system failures. States that do collect this information create their own definition of failure, which can range from sewage back-up to surface and/or ground water contamination (USEPA, 2002). Several studies have looked at the potential for septic systems to contaminate groundwater and drinking water sources, however none have looked at how failing systems compare to functioning systems in terms of water quality impacts (Cogger et al., 1984; Katz et al., 2011). In terms of GHG emissions, functioning septic systems have been estimated to release 0.22 tonne CO2- equivalents (CO2e) capita -1 year -1 to the environment (Diaz-Valbuena et al., 2011). However, data on GHG emissions from septic systems remains sparse with only a handful of papers addressing the subject and of those no study to-date has addressed the question of how failing, flooded septic systems compare to wellmaintained systems (Diaz-Valbuena et al., 2011; Kinnicutt et al., 1910; Truhlar et al., 2016). Based on a 2002 EPA report, the percentage of failed systems could range anywhere from 0.5% to 70% by state (US EPA, 2002). If these systems are failing at the upper end of the estimated rate, their contributions to GHG emissions as well as their effects on neighboring water bodies could be significant. It is important to determine the extent to which the lack of management and regulations on these systems can impact both air and water quality. Previous studies have overlooked the importance of microbial communities controlling GHG emissions from septic system leach field systems. Microorganisms involved in methane production (methanogens) and destruction (methanotrophs) can be studied using functional gene biomarkers mcra and pmoa, respectively, for quantification and characterization and have previously been found to correlate with CH 4 emissions from soils (Freitag et al., 2010; Lee et al., 2014). In particular, greater methane fluxes have been observed in flooded soils with corresponding higher abundances of mcra gene copies and transcripts (Ma et al., 2012). Flooded septic leach field systems are expected to display the same relationship and could have significantly different microbial populations as compared to well-maintained systems. The demand for low-cost, decentralized wastewater treatment could rise as growth in the suburban areas of New York s Hudson and Mohawk Valleys continues (Roberts, 2006). Thus, it is becoming increasingly imperative to determine the impact of these systems on the environment, with particular attention to how failing systems contribute to air and water pollution. The Hudson River Estuary Action Agenda (NYSDEC, 2010) includes goals for protecting water quality and reducing GHG emissions, septic systems have the potential to impact both of these areas and there is a significant lack of information surrounding these systems that needs to be addressed. Objectives This project builds on previous years work in which we found significant GHG emissions from septic systems relative to the surrounding landscape, particularly for failing systems, as well as the ubiquitous presence of

3 key functional biomarker genes involved in GHG cycling (Truhlar et al., 2016; Fernandez-Baca, unpublished). We further wanted to characterize the microbial communities populating these leach field systems and examine the impact of failing systems, specifically flooded systems, on both air and water quality. The objective of this project was to compare two septic leach field systems in four categories: (1) measured atmospheric methane (CH 4 ) fluxes; (2) measured CH 4 depth profile, (3) distribution and activity of key organisms involved in CH 4 cycling; (4) measured chemical oxygen demand (COD) and nutrient treatment (N, P). This research will aid in gaining a better understanding of how septic systems impact water quality and contribute to climate change. This work will emphasize the importance of effectively managing these systems to reduce air and water pollution. Results & Discussion Operation Two columns were operated under different conditions (Figure 1). Column A was operated under permanently Flooded conditions while Column B was operated under Well-Maintained conditions until August 10, From August 10 th, 2016 until February 19 th, 2017, Column B was operated under flooded conditions. After February 19 th Column B was reverted back to Well- Maintained conditions. where conditions are anaerobic. Column B showed no methane production at any depth before flooding. Ө Ө 1# 2# 3# 4# 5# CH 4 "Concentra8on"(uM)" Figure 2. Methane depth profile for (A) Column A and (B) Column B. Soil column schematic indicates location of port number with relation to soil column depth. CH 4 fluxes for Column A were significantly higher than Column B (p < 0.05) prior to flooding of B. Net emissions for Column B were on average 0 g CH 4 /day. p"<"0.0 *" Figure 3. CH 4 fluxes from Column A and B prior to flooding of B showed significant differences in CH 4 emissions. Upon flooding, Column B began to display increased CH 4 pore water concentrations with corresponding increases in surface CH 4 fluxes (Figures 4 and 5). With time, Column B s CH 4 depth profile began to resemble that of Column A. CH 4 "Concentra8on"(uM)" A# B# CH 4 "Concentra8on"(uM)" CH Concentra8on"(uM)" A" B" Figure 1. Schematic of soil column operation and setup. Column B has been operated under both well-maintained and flooded regimes. Methane Depth Profile and Fluxes Column A had greater CH 4 production in the soil profile compared to Column B (Figure 2) prior to flooding of Column B. Methane concentrations in Column A were highest near the wastewater inlet (Ports 2 and 3), Figure 4. Pore water CH 4 concentrations with depth for (A) Column A and (B) Column B after flooding Column B. Methane concentrations in Column B increased to levels comparable to Column A.

4 After prolonged flooding of Column B, there was no significant difference between emissions from Column A and Column B (Figure 5). below the soil surface near the wastewater inlet likely reflecting a niche for methanotrophs close to the subsurface source of methane. mcra, 10/10/16 pmoa, 10/10/16 Figure 5. Methane fluxes from Column A and B when both were operated in flooded conditions. Fluxes from Column B increased during this time. After returning Column B to normal, well-maintained operation, the pore water CH 4 concentrations began to decrease once again to pre-flooded levels. Column A continued to produce CH 4 near the inlet. CHConcentra8on"(uM)" CHConcentra8on"(uM)" A" B" Figure 6. Pore water CH 4 concentrations for (A) Column A and (B) Column B after returning Column B to 'well-maintained conditions. Column A remained flooded and producing CH 4 while Column B had a decrease in CH 4 production. Gene abundances Gene copies of pmoa and mcra were found in both systems (Figure XXX). DNA analysis of soils showed Column A had higher abundance of mcra compared to Column B immediately after flooding Column B, however under prolonged flooded conditions the abundance of mcra gene copies in Column C increased relative to Column A. Both columns had a peak in mcra gene abundance nearest Ports 2 and 3, where the influent wastewater is dosed. PmoA had greater gene copy levels in surface soils than mcra in both columns. Surprisingly, both columns showed greater abundance of pmoa (as well as mcra) Figure 7. Gene abundances for mcra and pmoa from two soil sample dates. Abundance of mcra increases in Column B with sustained flooding. PmoA is found throughout the column. Column B had greater COD removal on average (>90%) compared to A (30-65%) before flooding (Figure 8). COD%Percent%Removal%(%)% 120" 100" 80" 60" 40" 20" 0" Pre$Flood) 7/7/16% 8/3/16% mcra, 1/19/17 8/9/16% 8/26/16% Column"A" 8/29/16% 9/28/16% Column"B" 10/17/16% 1/19/17% pmoa, 1/19/17 Flooded) 2/3/17% Figure 8. COD removal was variable. Column B pre-flood had consistently higher COD removals than Column A but this difference was less prominent after both columns were flooded. Both columns had >90% phosphorus (P) and ammonium (NH 4 + ) removal suggesting nutrient removal was unaffected by flooding (Figures 9 and 10). Nitrate/nitrite were produced at low levels (0.3-2 mg-n/l) in the columns pore water and were reduced to below detection in effluent. This indicates that any production of nitrate and nitrite was low in these systems and likely not contributing to ground water contamination.

5 NHConcentraAon"(mg/L"as"N)" Figure 9. NH 4 removal in both columns was consistent. P"Concentra@on"(mg/L)" 80" 70" 60" 50" 40" 30" 20" 10" 0" 8" 7" 6" 0" 29,Jun" 29,Jun" Pre$Flood) 29,Jul" 28,Aug" Figure 10. P removal from both columns was >90% and was unaffected by flooding. Future shifts in DNA gene copies with time will be determined. As Column B reverts back to a wellmaintained system we expect to see a decrease in mcra gene abundance. We are further characterizing the microbial community using high-throughput sequencing of our soil samples. This additional data set will inform the groups of methanogens and methanotrophs responsible for the majority of CH 4 cycling and will shed light on the community shifts with soil depth and moisture. Understanding how the presence and abundance of biomarker genes are related to CH 4 cycling can elucidate what controls CH 4 emissions from these systems and inform future septic systems designs for better GHG mitigation strategies. Overall, the two columns performed more similarly in nutrient removal than in CH 4 cycling under both flooded and well-maintained conditions. Although effective nutrient removals (N and P) were achieved by both flooded and non-flooded leach field soils, COD removal was variable and did appear to be impacted by flooding. Methane was shown to be significantly higher in flooded systems both throughout 27,Sep" 27,Oct" 26,Nov" Post$Flood) Influent"A" Effluent"A" Influent"B" Effluent"B" Pre$Flood) 29,Jul" 28,Aug" 27,Sep" 26,Dec" 25,Jan" Post$Flood) 27,Oct" Influent"A" Effluent"A" Influent"B" Effluent"B" 26,Nov" the soil profile and in emissions from the soil surface. These results show that management of failing septic systems is vital for minimizing air and water pollution in New York state. Policy Implications This research shows that failing systems can negatively impact air quality and potentially also impair water quality. Policies should focus on enforcing maintenance of septic systems. Methods Flux measurements were done following a modified field flux chamber method from Molodovskya et al. (2011). Briefly, 5 ml gas samples are taken through a septa at the top of the cap every 10 minutes for 30 minutes and injected into pre-sealed 9 ml vial. The stored gas sample is analyzed via GC-FID on the same day. A linear regression was fit to the data to estimate flux from soil surface. The calculated flux was scaled by a typical leach field area a domestic household. Pore water samples were analyzed for ammonium, nitrate, nitrite, P and COD as well as dissolved methane. Nutrient concentrations were determined using published colorimetric assays ammonium (Bower and Holm-Hansen, 1980), nitrate/nitrite (Miranda et al., 2001), phosphorus (APHA, 2005) modified for a microplate reader. COD measurements were done using a CHEMetrics kit (Cat. No. K-7365). Dissolved methane was measured by injecting 5 ml of pore water into a pre-sealed 9 ml vial, shaking for 5 minutes and sampling the headspace for GC-FID analysis. Dissolved methane concentrations were back-calculated using Henry s Constant for methane partitioning in water. Soils were sampled periodically by drilling through the column and subsampling soil. DNA extractions were done using the MoBio RNA PowerSoil extraction kit (MoBio Laboratories, Carlsbad, CA). All reactions were run in triplicate using a total reaction volume of 25 µl. Each reaction was comprised of 2X iq SYBR Green Supermix (Bio-Rad, US), 17.5 pmol of primer, and 3 ul of template DNA (with concentrations of 10 ng/ul). Thermal cycling was conducted on an icycler IQ (Bio- Rad). Quantification analysis was carried out using Ct values from the icycler IQ software. Melt curve analyses were conducted on all products to ensure specificity.

6 Confirmation of products by Sanger sequencing showed amplification of targeted genes. Student Training Two undergraduate students were trained during the course of this project. Both were Juniors in Environmental Engineering and contributed directly to the data summarized here. Additional final reports related to water resource research are available at References APHA (2005) Standard methods for the examination of water and wastewater, 21st edn. American Public Health Association, Washington, DC Bower, C. E., & Holm-Hansen, T. (1980). A Salicylate Hypochlorite Method for Determining Ammonia in Seawater. Canadian Journal of Fisheries and Aquatic Sciences, 37(5), Cogger, C. G., & Carlile, B. L. (1984). Field Performance of Conventional and Alternative Septic Systems in Wet Soils1. Journal of Environment Quality, 13(1), 137. Diaz-Valbuena, L.R., H.L. Leverenz, C.D. Cappa, G. Tchobanoglous, W.R. Horwath, J.L. Darby Methane, carbon dioxide, and nitrous oxide emissions from Septic Tank Systems. Environmental Science & Technology 45(7): Fernandez-Baca, C., Pollard, J., Richardson, R. (2017). Methane and nutrient cycling: active processes in leach field soil systems. Manuscript in preparation. Freitag, T. E., Toet, S., Ineson, P., & Prosser, J. I. (2010). Links between methane flux and transcriptional activities of methanogens and methane oxidizers in a blanket peat bog. FEMS Microbiology Ecology, 73(1), Katz, B. G., Eberts, S. M., & Kauffman, L. J. (2011). Using Cl/Br ratios and other indicators to assess potential impacts on groundwater quality from septic systems: A review and examples from principal aquifers in the United States. Journal of Hydrology, 397(3-4), Kinnicutt, L. P., C.E.A. Winslow, R.W Pratt. (1910) Sewage Disposal. John Wiley and Sons: New York, Lee, H. J., Kim, S. Y., Kim, P. J., Madsen, E. L., & Jeon, C. O. (2014). Methane emission and dynamics of methanotrophic and methanogenic communities in a flooded rice field ecosystem. FEMS Microbiology Ecology, 88(1), Ma, K., Conrad, R., & Lu, Y. (2012). Responses of methanogen mcra genes and their transcripts to an alternate dry/wet cycle of paddy field soil. Applied and Environmental Microbiology, 78(2), Miranda, K. M., Espey, M. G., & Wink, D. A. (2001). A rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite. Nitric Oxide : Biology and Chemistry / Official Journal of the Nitric Oxide Society, 5(1), Molodovskaya, M., Warland, J., Richards, B. K., Öberg, G., & Steenhuis, T. S. (2011). Nitrous Oxide from Heterogeneous Agricultural Landscapes: Source Contribution Analysis by Eddy Covariance and Chambers. Soil Science Society of America Journal, 75(5), NYSDEC (2010) Hudson River Estuary Action Agenda pp. 55. Roberts, Sam. (2006). Hudson Valley becomes notable for its exurbanites, study finds. New York Times. October 19, Truhlar, A. M., Rahm, B. G., Brooks, R. A., Nadeau, S. A., Makarsky, E. T., & Walter, M. T. (2016). Greenhouse Gas Emissions from Septic Systems in New York State. Journal of Environment Quality, 45(4), 1153.

7 USEPA Onsite Wastewater Treatment Systems Manual. Environmental Protection Agency Report EPA625/R-00/008, 175pp. USEPA Decentralized Wastewater Treatment Can Be Green and Sustainable. USEPA. < /MOU-Green-Paper v2.pdf.> < USEPA Decentralized Wastewater Management Program Highlights, EPA-832-R USEPA. US EPA (last updated Oct. 2013) Office of Wastewater Management Decentralized Program