Estrogens are known endocrine disruptor compounds (EDC) that can affect aquatic life at concentrations as low as 0.35 ng/L. A main source of aquatic estrogen contamination is wastewater treatment plant (WWTPs) effluents. Estrogens enter the WWTP through normal human water use. In order of their EDC potency these estrogens are 17á-ethynyl estradiol (EE2, a synthetic estrogen), 17â- estradiol (E2), estrone (E1) and estriol (E3). Biological degradation is a primary estrogen removal mechanism at WWTPs and EE2 is degraded much slower than E1 and E2. Prior work by the research team found that heterotrophic bacteria play a major role in estrogen biodegradation even when grown on a wide range of organic substrates. Laboratory experiments with three bioreactors fed synthetic feed showed that EE2, E1 and E2 degradation kinetics varied among reactors configured for anaerobic/aerobic sequenced treatment, which selects for phosphorus accumulation organism; anoxic/aerobic sequenced treatment, which selects for facultative organisms; and aerobic treatment. The anaerobic and anoxic selector designs are also important for biological nutrient removal (BNR) systems, commonly used today. Critical questions for this research are: (1) will similar relative degradation kinetics for the different microbial populations (anaerobic>aerobic>anoxic) occur when treating actual municipal wastewater versus synthetic, (2) how will microbial populations compare within the selector designs, and (3) can EE2-degrading bacteria be identified for study in pure culture. Parallel bench scale reactors similar to those described above for the three configurations treating municipal primary effluent will be integrated with mechanistic modeling and advanced molecular and microbial techniques to address biodegradation kinetics of estrogens at relevant ng/L concentrations.

Specific research goals include: (1) Evaluate the effect of selector/BNR process designs on estrogen removal performance; (2) compare the EE2, E1, and E2 degradation kinetics in the different reactor configurations; (3) characterize the microbial populations for the different selector designs through terminal restriction fragment length polymorphism (T-RFLP), (4) determine if the fraction of estrogen-degrading biomass is higher for the bioreactors with higher specific estrogen degradation rates, and (5) evaluate estrogen degradation ability of microbial isolates obtained from the study bioreactors (including anaerobic and anoxic selectors). The results will be incorporated into a comprehensive estrogen/activated sludge model based on the International Water Association ASM1 and ASM2d models. The estrogen/ASM1 model has been completed by us by applying the industry GPS-X software. Similar work will be done with the ASM2d model, which includes enhanced biological phosphorus removal. The models include free and conjugated forms of E1, E2 and EE2, deconjugation and biodegradation kinetics, possible production of E1 from E2 degradation, and liquid-solids partitioning of estrogens.

The intellectual merit of the project is its transformational approach, which integrates modeling the fate of a micropollutant in a biological process with a fundamental co-substrate mechanism, bioselector design effects, and microbial composition based on molecular methods. A second potential far reaching benefit will be the development of pure cultures capable of EE2 degradation under condition similar to WWTP for future kinetic and genetic studies, and development of qPCR primer sets for monitoring select EE2-degrading heterotrophs in WWTP facilities.

The broader impacts of the project are benefits to society by providing a basis to optimize WWTP biotreatment design to minimize estrogen release to the environment. Direct educational benefits include the training of graduate students, and participation of undergraduate researchers. Increased participation by underrepresented groups will be realized through established University of Washington programs and continued partnership with the UW, College of Engineering Office of Diversity.

Project Report

Municipal wastewater treatment plants (WWTPs) receive a broad spectrum of organic, nitrogenous and phosphorus-containing compounds chemicals and in most cases employ activated sludge treatment processes for their removal to very high efficiencies (often at greater than 90%) before discharge to surface waters. More recently a wide range of compounds termed micropollutants, which are present at extremely low concentration (nanogram per liter (ng/L) or microgram per liter (μg/L), have become a concern because of their potential harm to aquatic life and threat to human water supplies. These compound arrive at WWTPs due to human use of pharmaceuticals, personal care products, and household cleaning chemicals and release of natural hormones. A number of these compounds disrupt endocrine system activity, which is important for regulating growth, metabolism, behavior, and reproduction in animals and aquatic life. Of these, estrogen compounds are the most potent endocrine disrupting compounds (EDCs); the natural estrogen hormones (E1, and E2) and the synthetic estrogen compound used in birth control pills, EE2 and its conjugates (EE2-3G, and EE2-3S) are shown in Figure 1. Municipal WWTP effluents and animal feedlots are the major sources of estrogen discharge to surface waters. There are numerous results from field studies showing significant impacts of EDCs on fish reproduction system after WWTP discharge outfalls. Predicted no-effect concentrations of E1, E2, and EE2 in surface waters are 6, 2, and 0.1 ng/L, respectively, well below the concentrations often found in WWTP effluents. To put these values in perspective, a commonly accepted good WWTP effluent concentration for ammonia-nitrogen is 1.0 mg/L or 1,000,000 ng/L. Fortunately estrogen compounds are removed in WWTP activated sludge systems, primarily via biodegradation by heterotrophic bacteria, but the degradation rate of EE2 can be 1/5th to 1/3rd that of the other estrogens. The biodegradation and fate of EE2 in activated sludge treatment was thus the major focus of this research due to its lower degradation rate and greater effect at low concentrations in surface waters. Reported influent concentrations of EE2 to WWTPs have ranged from below detection to 330 ng/L and removal efficiencies have varied widely, from 30 to 90 percent. A major goal of this research was to find out what factors were most important for EE2 biodegradation and how knowledge of such factors could be used to provide lower treated effluent concentrations to possibly avoid the use of more expensive treatment alternatives. A basic hypothesis of the research was that different biotreatment design known to select for different bacteria also would result in different levels of EE2-biodegraders and different biodegradation biokinetics and treatment efficiency. In addition the degradation kinetics of converting the conjugated form of estrogen compounds in the influent wastewater to the free estrogen forms was determined. Many activated sludge process configurations are used in wastewater treatment but generally involve the elements of the common bioselector designs depicted in Figure 2; initial feeding zones that may be aerobic, anoxic, or anaerobic. The anoxic and anaerobic bioselector designs are commonly used for nitrogen and phosphorus removal, respectively, to limit the discharge of nutrients and minimize eutrophication in surface waters. The research involved experiments with activated sludge bioreactor configurations in laboratory studies and in field studies treating municipal wastewater to evaluate the effect of various selector designs on estrogen removal at ng/L concentrations. The estrogen biodegradation kinetics were studied and a mechanistic model was successfully constructed and calibrated to predict WWTP effluent estrogen concentration as a function of the activated sludge process configuration and bioselector design, influent wastewater characteristics, and operating conditions. Intellectual Merit: This was the first study to systematically evaluate the effect of bioselector designs on estrogen removal efficiency. It found that the estrogen removal was not hindered by using important biological nutrient removal designs and identified bioselector designs that favored higher estrogen biodegradation kinetics and better removal efficiency. It identified biological treatment processes that would provide more optimal estrogen removal efficiency, such as staged aerobic reactors and oxidation ditch processes. It also identified important wastewater parameters that affect estrogen removal efficiency, which helped explain the variability of reported WWTP performance. The mathematical model provides a powerful tool to evaluate estrogen removal efficiency for different wastewaters and designs. An example of general results from model simulations is shown in Figure 3. High desired estrogen removal efficiency of 90% by biodegradations is only possible with batch fed or stage aeration tanks and with a biokinetic coefficient value of greater than 10 L/g COD-d. Broad impacts: The research led to more cost effective treatment for removal of a most important endocrine disruptor compound, advancing understanding and approaches to assess micropollutant removal, the development of fundamental information and tools for education, technology transfer to designers, and the advancement of education and research experience for students from underrepresented areas in the environmental engineering profession.

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