X-ray absorption near edge structure spectroscopy reveals phosphate minerals at surface and agronomic sampling depths in agricultural Ultisols saturated with legacy phosphorus.

Legacy phosphorus (P) soils have received excessive P inputs from historic manure and fertilizer applications and present unique management challenges for protecting water quality as soil P saturation leads to increased soluble P to waterways. We used P K-edge X-ray absorption near edge structure (XANES) spectroscopy to identify and quantify the dominant P minerals in four representative legacy P soils under conventional till and no-till management in Maryland, USA. Various measures of extractable soil P, including water-extractable P (20.6-54.1 mg kg-1 at 1:10 soil-to-water ratio; 52.7-132.2 mg kg-1 at 1:100 soil-to-water ratio), plant available P extracted with Mehlich 3 (692-1139 mg kg-1), and Mehlich 3P saturation ratio (0.54-1.37), were above the environmental threshold values, suggesting the accumulation of legacy P in soils. The quantification of dominant P minerals may provide insights into the potential of legacy P soils to contribute to P release for crop use and soluble P losses. Linear combination fits of XANES spectra identified the presence of four phosphate mineral groups, consisting of (i) calcium-phosphate minerals (11-59%) in the form of fluorapatite, ß-tricalcium phosphate, and brushite, followed by (ii) iron-phosphate minerals (12-49%) in the form of ludlamite, heterosite, P sorbed to ferrihydrite, and amorphous iron phosphates, (iii) aluminum-phosphate minerals (15-33%) in the form of wavellite and P sorbed to aluminum hydroxide, and (iv) other phosphate minerals (5-35%) in the form of copper-phosphate (cornetite, 5-18%) and manganese-phosphate (hureaulite, 25-35%). Organic P consisting of phytic acid was found in most soils (13-24%) and was more pronounced in the surface layer of no-till (21-24%) than in tilled (16%) fields. Of the P forms identified with XANES, we conclude that P sorbed to Fe and Al, and Ca-P in the form of brushite and ß-tricalcium phosphate will likely readily contribute to the soil WEP pool as the soil solution P is depleted by crop uptake and lost via runoff and leaching.

Molecular characterization of dissolved organic nitrogen and phosphorus in agricultural runoff and surface waters.

Agricultural runoff is a significant contributor to nitrogen (N) and phosphorus (P) pollution in water bodies. Limited information is available about the molecular characteristics of the dissolved organic N (DON) and P (DOP) species in the agricultural runoff and surface waters. We employed Fourier Transform-Ion Cyclotron Resonance-Mass Spectrometry (FT-ICR-MS) to investigate the changes in the molecular characteristics of DON and DOP at three watershed positions (upstream water, runoff from agricultural fields, and downstream waters). Across three watershed locations, more-bioavailable compounds (such as amino sugars, carbohydrates, lipids, and proteins) accounted for <5% of DON and 4-31% of DOP molecules, whereas less-bioavailable compounds (such as lignin, tannins, condensed hydrocarbons, and unsaturated hydrocarbons) were >95% of DON and 69-96% of DOP. Of the dissolved organic matter, runoff waters from agricultural fields contained the greatest proportion of DON formulas (20-25%) than upstream (18%) and downstream (13-14%) waters, indicating the presence of a greater diversity of DON species in the runoff. Various nutrient sources present in agricultural fields such as crop residues, soil organic matter, and transformed fertilizers likely contributed to the diverse composition of DON and DOP in the runoff, which were likely altered as the surface water traversed along the flow pathways in the watershed. The presence of more-bioavailable molecules detected in upstream compared to agricultural runoff and downstream waters suggests that photochemical and/or microbial processes likely altered the characteristics of DON and DOP compounds. The findings of this study increase our understanding of DON and DOP compounds lability and transformations in runoff and surface waters , which may be useful in quantifying the contribution of organic N and P sources to water quality impairment in aquatic ecosystems.

Residential catchments to coastal waters: Forms, fluxes, and mechanisms of phosphorus transport.

Controlling phosphorus (P) loss from land to water bodies is of immense scientific and societal interest and scrutiny. We investigated P forms in a longitudinal gradient in three typical urban junctions: stormwater from a residential catchment, pond discharges from a stormwater retention pond, and 13 coastal waters (rivers and estuary). Concentrations of total P (TP) were 122.7 ± 99.1 µg/L in the stormwater, 89.7 ± 35.8 µg/L in the pond discharges, and 212.1 ± 51.2 µg/L in 13 coastal water sites. Lower P concentrations in pond discharges reflect P attenuation in the stormwater pond, and higher P concentrations in surface waters are likely attributed to the additional contributing P sources in the watershed. Dissolved reactive P (DRP) was 38% of TP load in stormwater and 46% of TP concentrations in surface water sites, whereas particulate unreactive P (PUP) was 52% of TP load in pond discharges. The first-flush strength of P forms in the stormwater indicated the dominance of particulate P over dissolved P. More particulate P was transported in the early stages of storms due to the runoff of P associated with sediment, plant materials, and built up on impervious surfaces. Whereas more dissolved P was transported in the later stages of storms likely due to the flushing of P, as exacerbated by greater runoff amounts, from the landscape sources, i.e., grass clippings, tree leaves, and soil. In the pond discharges, DRP was a minor form suggesting its utilization by bacteria and algae in the pond. The high concentration and proportion of DRP in surface waters suggest an abundance of bioavailable P in urban waters. These results imply that treatment designs in urban areas should consider ways to remove P in urban landscapes focusing on attenuating P before the initiation of runoff and discharge to surface waters to protect downstream water quality.

Wet season nitrogen export from a residential stormwater pond.

Stormwater runoff is recognized as a cause of water quality degradation because it may carry nitrogen (N) and other pollutants to aquatic ecosystems. Stormwater ponds are a stormwater control measure often used to manage stormwater runoff by holding a permanent pool of water, which reduces the peak flow, magnitude of runoff volume, and concentrations of nutrients and pollutants. We instrumented the outlet of a stormwater pond in an urban residential neighbourhood in Florida, United States to (1) investigate the concentration and composition of N forms during the summer rainy season (May to September 2016), and (2) determine the bioavailability of organic N in the stormwater pond with a bioassay experiment. A total of 144 outflow water samples over 13 storm events were collected at the outlet of the stormwater pond that collects runoff from the residential catchment. Samples were analysed for various inorganic N [ammonium (NH4-N), nitrate (NO3-N)], and organic N forms [dissolved organic nitrogen (DON), and particulate organic nitrogen (PON)]. Flow-weighted mean concentration of total N (TN) in pond outflow for all collected storm events was 1.3±1.42 mg L-1, with DON as the dominant form (78%), followed by PON and NO3-N (each at 8%), and NH4-N (6%). In the bioassay experiment, organic N (DON+PON) was significantly decreased by 25-28% after 5 days of incubation, suggesting that a portion of the DON carried from the pond outflow to receiving water bodies may be bioavailable. These results suggest that efforts to mitigate stormwater N outflows from urban ponds should incorporate both inorganic and organic N in management plans.

Composition of nitrogen in urban residential stormwater runoff: Concentrations, loads, and source characterization of nitrate and organic nitrogen.

Stormwater runoff is a leading cause of nitrogen (N) transport to water bodies and hence one means of water quality deterioration. Stormwater runoff was monitored in an urban residential catchment (drainage area: 3.89 hectares) in Florida, United States to investigate the concentrations, forms, and sources of N. Runoff samples were collected over 22 storm events (May to September 2016) at the end of a stormwater pipe that delivers runoff from the catchment to the stormwater pond. Various N forms such as ammonium (NH4-N), nitrate (NOx-N), dissolved organic nitrogen (DON), and particulate organic nitrogen (PON) were determined and isotopic characterization tools were used to infer sources of NO3-N and PON in collected runoff samples. The DON was the dominant N form in runoff (47%) followed by PON (22%), NOx-N (17%), and NH4-N (14%). Three N forms (NOx-N, NH4-N, and PON) were positively correlated with total rainfall and antecedent dry period, suggesting longer dry periods and higher rainfall amounts are significant drivers for transport of these N forms. Whereas DON was positively correlated to only rainfall intensity indicating that higher intensity rain may flush out DON from soils and cause leaching of DON from particulates present in the residential catchment. We discovered, using stable isotopes of NO3-, a shifting pattern of NO3- sources from atmospheric deposition to inorganic N fertilizers in events with higher and longer duration of rainfall. The stable isotopes of PON confirmed that plant material (oak detritus, grass clippings) were the primary sources of PON in stormwater runoff. Our results demonstrate that practices targeting both inorganic and organic N are needed to control N transport from residential catchments to receiving waters.

Stormwater runoff driven phosphorus transport in an urban residential catchment: Implications for protecting water quality in urban watersheds.

Increased stormwater runoff in urban watersheds is a leading cause of nonpoint phosphorus (P) pollution. We investigated the concentrations, forms, and temporal trends of P in stormwater runoff from a residential catchment (31 low-density residential homes; 0.11 km2 drainage area) in Florida. Unfiltered runoff samples were collected at 5 min intervals over 29 storm events with an autosampler installed at the stormwater outflow pipe. Mean concentrations of orthophosphate (PO4-P) were 0.18 ± 0.065 mg/L and total P (TP) were 0.28 ± 0.062 mg/L in all runoff samples. The PO4-P was the dominant form in >90% of storm events and other-P (combination of organic P and particulate P) was dominant after a longer antecedent dry period. We hypothesize that in the stormwater runoff, PO4-P likely originated from soluble and desorbed pool of eroded soil and other-P likely originated from decomposing plant materials i.e. leaves and grass clippings and eroded soil. We found that the runoff was co-limited with nitrogen (N) and P in 34% of storm events and only N limited in 66% of storm events, implicating that management strategies focusing on curtailing both P and N transport would be more effective than focussing on only N or P in protecting water quality in residential catchments.

Micropollutants in groundwater from septic systems: Transformations, transport mechanisms, and human health risk assessment.

Septic systems may contribute micropollutants to shallow groundwater and surface water. We constructed two in situ conventional drainfields (drip dispersal and gravel trench) and an advanced drainfield of septic systems to investigate the fate and transport of micropollutants to shallow groundwater. Unsaturated soil-water and groundwater samples were collected, over 32 sampling events (January 2013 to June 2014), from the drainfields (0.31-1.07 m deep) and piezometers (3.1-3.4 m deep). In addition to soil-water and groundwater, effluent samples collected from the septic tank were also analyzed for 20 selected micropollutants, including wastewater markers, hormones, pharmaceuticals and personal care products (PPCPs), a plasticizer, and their transformation products. The removal efficiencies of micropollutants from septic tank effluent to groundwater were similar among three septic systems and were 51-89% for sucralose and 53->99% for other micropollutants. Even with high removal rates within the drainfields, six PPCPs and sucralose with concentrations ranging from <0.3 to 154 ng/L and 121 to 32,000 ng/L reached shallow groundwater, respectively. The human health risk assessment showed that the risk to human health due to consumption of groundwater is negligible for the micropollutants monitored in the study. A better understanding of ecotoxicological effects of micropollutant mixtures from septic systems to ecosystem and human health is warranted for the long-term sustainability of septic systems.

Managing urban runoff in residential neighborhoods: Nitrogen and phosphorus in lawn irrigation driven runoff.

Sources and mechanisms of nutrient transport in lawn irrigation driven surface runoff are largely unknown. We investigated the transport of nitrogen (N) and phosphorus (P) in lawn irrigation driven surface runoff from a residential neighborhood (28 ha) of 56% impervious and 44% pervious areas. Pervious areas encompassing turfgrass (lawns) in the neighborhood were irrigated with the reclaimed water in common areas during the evening to late night and with the municipal water in homeowner's lawns during the morning. The stormwater outlet pipe draining the residential neighborhood was instrumented with a flow meter and Hach autosampler. Water samples were collected every 1-h and triple composite samples were obtained at 3-h intervals during an intensive sampling period of 1-week. Mean concentrations, over 56 sampling events, of total N (TN) and total P (TP) in surface runoff at the outlet pipe were 10.9±6.34 and 1.3±1.03 mg L-1, respectively. Of TN, the proportion of nitrate-N was 58% and other-N was 42%, whereas of TP, orthophosphate-P was 75% and other-P was 25%. Flow and nutrient (N and P) concentrations were lowest from 6:00 a.m. to noon, which corresponded with the use of municipal water and highest from 6:00 p.m. to midnight, which corresponded with the use of reclaimed water. This data suggests that N and P originating in lawn irrigation driven surface runoff from residential catchments is an important contributor of nutrients in surface waters.

Sources and mechanisms of nitrate and orthophosphate transport in urban stormwater runoff from residential catchments.

Nutrients export from residential catchments contributes to water quality impairment in urban water bodies. We investigated the concentrations, transport mechanisms, and sources of nitrate-nitrogen (NO3-N) and orthophosphate-phosphorus (PO4-P) in urban stormwater runoff generated in residential catchments in Tampa Bay, Florida, United States. Street runoff samples, collected over 21 storm events, were supplemented with rainfall and roof runoff samples from six representative residential catchments. Samples were analyzed for N and P forms, N and oxygen (O) isotopes of nitrate (δ18O-NO3- and δ15N-NO3-), and δ18O and hydrogen (δD) isotopes of water (H2O). We found that the main NO3-N source in street runoff was atmospheric deposition (range: 35-64%), followed by chemical N fertilizers (range: 1-39%), and soil and organic N (range: 7-33%), whereas PO4-P in the street runoff likely originated from erosion of soil particles and mineralization from organic materials (leaves, grass clippings). The variability in the sources and concentrations of NO3-N and PO4-P across catchments is attributed to different development designs and patterns, use of various fill materials during land development, and landscaping practices. This data can be useful to develop strategies to offset the impacts of urban development (e.g., designs and patterns resulting in variable impervious areas) and management (e.g., fertilizer use, landscaping practices) on NO3-N and PO4-P transport in urban residential catchments.

Rainfall-runoff of anthropogenic waste indicators from agricultural fields applied with municipal biosolids.

The presence of anthropogenic contaminants such as antimicrobials, flame-retardants, and plasticizers in runoff from agricultural fields applied with municipal biosolids may pose a potential threat to the environment. This study assesses the potential for rainfall-induced runoff of 69 anthropogenic waste indicators (AWIs), widely found in household and industrial products, from biosolids amended field plots. The agricultural field containing the test plots was treated with biosolids for the first time immediately prior to this study. AWIs present in soil and biosolids were isolated by continuous liquid-liquid extraction and analyzed by full-scan gas chromatography/mass spectrometry. Results for 18 AWIs were not evaluated due to their presence in field blank QC samples, and another 34 did not have sufficient detection frequency in samples to analyze trends in data. A total of 17 AWIs, including 4-nonylphenol, triclosan, and tris(2-butoxyethyl)phosphate, were present in runoff with acceptable data quality and frequency for subsequent interpretation. Runoff samples were collected 5days prior to and 1, 9, and 35days after biosolids application. Of the 17 AWIs considered, 14 were not detected in pre-application samples, or their concentrations were much smaller than in the sample collected one day after application. A range of trends was observed for individual AWI concentrations (typically from 0.1 to 10µg/L) over the course of the study, depending on the combination of partitioning and degradation mechanisms affecting each compound most strongly. Overall, these results indicate that rainfall can mobilize anthropogenic contaminants from biosolids-amended agricultural fields, directly to surface waters and redistribute them to terrestrial sites away from the point of application via runoff. For 14 of 17 compounds examined, the potential for runoff remobilization during rainstorms persists even after three 100-year rainstorm-equivalent simulations and the passage of a month.

Septic systems as hot-spots of pollutants in the environment: Fate and mass balance of micropollutants in septic drainfields.

Septic systems, a common type of onsite wastewater treatment systems, can be an important source of micropollutants in the environment. We investigated the fate and mass balance of 17 micropollutants, including wastewater markers, hormones, pharmaceuticals and personal care products (PPCPs) in the drainfield of a septic system. Drainfields were replicated in lysimeters (1.5m length, 0.9m width, 0.9m height) and managed similar to the field practice. In each lysimeter, a drip line dispersed 9L of septic tank effluent (STE) per day (equivalent to 32.29L/m(2) per day). Fourteen micropollutants in the STE and 12 in the leachate from drainfields were detected over eight months. Concentrations of most micropollutants in the leachate were low (<200ng/L) when compared to STE because >85% of the added micropollutants except for sucralose were attenuated in the drainfield. We discovered that sorption was the key mechanism for retention of carbamazepine and partially for sulfamethoxazole, whereas microbial degradation likely attenuated acetaminophen in the drainfield. This data suggests that sorption and microbial degradation limited transport of micropollutants from the drainfields. However, the leaching of small amounts of micropollutants indicate that septic systems are hot-spots of micropollutants in the environment and a better understanding of micropollutants in septic systems is needed to protect groundwater quality.

δ(15)N and δ(18)O Reveal the Sources of Nitrate-Nitrogen in Urban Residential Stormwater Runoff.

Nitrogen (N) sources are widely distributed in the complex urban environment. High-resolution data elucidating N sources in the residential catchments are not available. We used stable isotopes of N and oxygen (O) of nitrate (δ(18)O-NO3(-) and δ(15)N-NO3(-)) along with δ(18)O and hydrogen (δD) of water (H2O) to understand the sources and transformations of N in residential stormwater runoff. Stormwater runoff samples were collected over 25 stormwater events at 5 min intervals using an autosampler installed at the residential catchment outlet pipe that drained 31 low-density homes with a total drainage area of 0.11 km(2). Bayesian mixing model results indicated that atmospheric deposition (range 43-71%) and chemical N fertilizers (range <1-49%) were the dominant NO3-N sources in the stormwater runoff and that there was a continuum of source changes during the stormwater events. Further, the NO3-N transport in the stormwater runoff from the residential catchment was driven by mixing of multiple sources and biotic (i.e., nitrification) processes. This work suggests that a better understanding of N transport and sources is needed to reduce N export and improve water quality in urban water systems.

Subcellular distribution and chemical forms of cadmium in a dark septate endophyte (DSE), Exophiala pisciphila.

Our objective was to understand the cadmium (Cd) tolerance mechanisms by investigating the subcellular distribution, chemical forms of Cd and adsorptive groups in the mycelia of Exophiala pisciphila. We grew E. pisciphila in the liquid media with increasing Cd concentrations (0, 25, 50, 100, 200, and 400 mg L(-1)). Increased Cd in the media caused a proportional increase in the Cd uptake by E. pisciphila. Subcellular distribution indicated that 81 to 97% of Cd was associated with the cell walls. The largest amount and proportion (45-86%) of Cd was extracted with 2% acetic acid, and a concentration-dependent extraction was observed, both of which suggest that Cd-phosphate complexes were the major chemical form in E. pisciphila. A large distribution of phosphate and Cd on the mycelia surface was observed by scanning electron microscopy-energy dispersive spectrometer (SEM-EDS). The precipitates associated with the mycelia were observed to contain Cd by transmission electron microscopy-energy dispersive X-ray spectroscopy (TEM-EDX). Fourier transform infrared (FTIR) identified that hydroxyl, amine, carboxyl, and phosphate groups were responsible for binding Cd. We conclude that Cd associated with cell walls and integrated with phosphate might be responsible for the tolerance of E. pisciphila to Cd.

Tolerance and antioxidant response of a dark septate endophyte (DSE), Exophiala pisciphila, to cadmium stress.

The growth, oxidative damage and antioxidant response of Exophiala pisciphila ACCC32496, a dark septate endophyte isolated from an abandoned lead-zinc mining area, were measured at cadmium (Cd) concentrations of 0, 25, 50, 100, 200 and 400 mg L(-1). The EC50 values of E. pisciphila ACCC32496 to Cd were 332.2 mg L(-1) after 30 days on solid medium and 111.2 mg L(-1) after 7 days in liquid medium. Cd stress markedly stimulated the production of superoxide anion, H2O2 and malondialdehyde in the fungal mycelia. The activities of superoxide dismutase and catalase reached their maxima at 100 mg L(-1) Cd. The glutathione and non-protein thiol contents, along with the total antioxidant capability, reached their maxima at 50 mg L(-1) Cd. Low Cd concentrations induced a noticeable increase in antioxidant defense, while high Cd concentrations decreased the antioxidant defense.

Steroid hormone runoff from agricultural test plots applied with municipal biosolids.

The potential presence of steroid hormones in runoff from sites where biosolids have been used as agricultural fertilizers is an environmental concern. A study was conducted to assess the potential for runoff of seventeen different hormones and two sterols, including androgens, estrogens, and progestogens from agricultural test plots. The field containing the test plots had been applied with biosolids for the first time immediately prior to this study. Target compounds were isolated by solid-phase extraction (water samples) and pressurized solvent extraction (solid samples), derivatized, and analyzed by gas chromatography-tandem mass spectrometry. Runoff samples collected prior to biosolids application had low concentrations of two hormones (estrone <0.8 to 2.23 ng L(-1) and androstenedione <0.8 to 1.54 ng L(-1)) and cholesterol (22.5 ± 3.8 µg L(-1)). In contrast, significantly higher concentrations of multiple estrogens (<0.8 to 25.0 ng L(-1)), androgens (<2 to 216 ng L(-1)), and progesterone (<8 to 98.9 ng L(-1)) were observed in runoff samples taken 1, 8, and 35 days after biosolids application. A significant positive correlation was observed between antecedent rainfall amount and hormone mass loads (runoff). Hormones in runoff were primarily present in the dissolved phase (<0.7-µm GF filter), and, to a lesser extent bound to the suspended-particle phase. Overall, these results indicate that rainfall can mobilize hormones from biosolids-amended agricultural fields, directly to surface waters or redistributed to terrestrial sites away from the point of application via runoff. Although concentrations decrease over time, 35 days is insufficient for complete degradation of hormones in soil at this site.

Testosterone-mineralizing culture enriched from swine manure: characterization of degradation pathways and microbial community composition.

Environmental releases and fate of steroid sex hormones from livestock and wastewater treatment plants are of increasing regulatory concern. Despite the detection of these hormones in manures, biosolids, and the environment, little attention has been paid to characterization of fecal bacteria capable of hormone degradation. The enrichments of (swine) manure-borne bacteria capable of aerobic testosterone degradation were prepared and the testosterone mineralization pathway was elucidated. Six DNA sequences of bacteria from the Proteobacteria phylum distributed among the genera Acinetobacter, Brevundimonas, Comamonas, Sphingomonas, Stenotrophomonas, and Rhodobacter were identified in a testosterone-degrading enriched culture with testosterone as the sole carbon source. Three degradation products of testosterone were identified as androstenedione, androstadienedione, and dehydrotestosterone using commercially available reference standards, liquid chromatography-UV diode array detection, and liquid chromatography-time-of-flight mass spectrometry (LC-TOF/MS). Three additional degradation products of testosterone were tentatively identified as 9α-hydroxytestosterone, 9α-hydroxyandrostadienedione or 3-hydroxy-9,10-secoandrosta-1,3,5(10)-triene-9,17-dione, and 9α-hydroxydehydrotestosterone or 9α-hydroxyandrostenedione using LC-TOF/MS. When (14)C-testosterone was introduced to the enriched culture, 49-68% of the added (14)C-testosterone was mineralized to (14)CO(2) within 8 days of incubation. The mineralization of (14)C-testosterone followed pseudo-first-order reaction kinetics in the enriched culture with half-lives (t(1/2)) of 10-143 h. This work suggests that Proteobacteria play an important environmental role in degradation of steroid sex hormones and that androgens have the potential to be mineralized during aerobic manure treatment or after land application to agricultural fields by manure-borne bacteria.

Degradation kinetics of testosterone by manure-borne bacteria: influence of temperature, pH, glucose amendments, and dissolved oxygen.

Land application of manure may contribute endocrine disrupting compounds (EDCs) such as steroid hormones to the environment. Little attention has been paid to the potential for degradation of steroid hormones by manure-borne bacteria and their degradation kinetics and pathways. In a laboratory study, the potential for biodegradation of testosterone, 17beta-estradiol (E2) and progesterone by swine (Sus scrofa) manure-borne bacteria was examined. In addition, the impact of temperature, pH (6, 7, and 7.5), glucose amendments (0, 3, and 22 mmol L(-1)), and presence of oxygen on testosterone degradation kinetics was determined. Testosterone, 17beta-estradiol and progesterone were biodegraded within 25 h of reaction initiation under aerobic conditions. The degradation of testosterone followed pseudo first-order and zero-order reaction kinetics under aerobic and anaerobic conditions, respectively, in tryptic soy broth (TSB) pre-enriched systems. The half-life (t1/2) for the degradation of testosterone under anaerobic conditions was six times longer than aerobic conditions. Testosterone degradation was found to significantly increase (- 17%) when incubated at 37 degrees C vs. 22 degrees C. The impact of pH (t1/2 ranged from 4.4-4.9 h) and glucose amendments (t1/2 ranged from 4.6-5.1 h) on the testosterone degradation rate were found to be small. Testosterone was transformed to dehydrotestosterone (DHT) (major degradation product), androstenedione (AD), and androstadienedione (ADD) under aerobic conditions as revealed by liquid chromatography-time-of-flight mass spectrometry (LC/TOF-MS). These results indicate that testosterone is rapidly degraded by manure-borne bacteria under a wide range of environmentally relevant conditions. However, the formed degradation products are still of potential concern due to their endocrine disrupting potential.