Influence of Agrochemical Mixtures on Treatment Wetland Ecosystems Services

INFLUENCE OF AGROCHEMICAL MIXTURES ON TREATMENT WETLAND ECOSYSTEMS SERVICES

Sponsoring Institution

National Institute of Food and Agriculture

Project Status

ACTIVE

Funding Source

AFRI COMPETITIVE GRANT

Reporting Frequency

Annual

Accession No.

1019018

Grant No.

2019-67019-29487

Cumulative Award Amt.

$499,999.00

Proposal No.

2018-06863

Multistate No.

(N/A)

Project Start Date

May 1, 2019

Project End Date

Apr 30, 2025

Grant Year

2019

Program Code

[A1401]- Foundational Program: Soil Health

Recipient Organization
UNIVERSITY OF NEBRASKA
(N/A)
LINCOLN,NE 68583

Performing Department
Civil Engineering

Non Technical Summary
Natural and constructed wetlands are now used extensively across the United States as a means for mitigating nitrate losses to both surface water and groundwater. However, property and management requirements limit wetland use. Floating treatment wetlands, floating macrophyte mats, positioned in adjacent water storage ponds have the potential to a wide range of contaminants (i.e., nitrate, phosphate, antibiotics) from source water without the limitations of property and management requirements. While the use of floating treatment wetlands as a treatment approach for nitrate in run-off is an emerging practice, nitrate-N is not the sole constituent coming from agricultural fields. Other biologically active contaminants regularly co-occur with nitrate and many can affect the efficiency of nitrate removal. This project will evaluate the discrete and combined effects of specific veterinary antibiotics and an increasingly used nitrification inhibitor (nitrapyrin) to nitrogen transformation and its potential effect on nitrate-N treatment efficiency in saturated sediments and floating treatment wetlands. Specific research objectives of our project are to: 1) simulate and quantify the combined effects of antibiotics and a commercial nitrification inhibitor on the nitrogen cycle in floating treatment wetlands; and 2) determine the overall effect of trace levels of antibiotics and nitrification inhibitors on the nitrate-N removal potential of floating treatment wetlands receiving contaminated agricultural run-off. We will first conduct microcosm incubations at a USGS research laboratory to establish the concentration levels where nitrapyrin and antibiotic mixtures affect nitrate transformation. Nitrogen and antibiotic transformation, and nitrapyrin interactions will be assessed in two sets of mesocosm floating treatment wetland experiments using methods from past successful simulations. The transformation and fate of selected veterinary antibiotics will also be tracked in the mesocosms. Findings will provide new insight into whether residues of nitrification inhibitors and veterinary antibiotics in these environments will affect proposed mitigation strategies for controlling nitrogen losses from fertilized crops and managing nitrate contamination of ground and surface water.The interdisciplinary team assembled for this project provides a rare opportunity to evaluate a complex ecosystem from an integrated microbiology, biogeochemistry, analytical hydrochemistry, and environmental engineering perspective. Collaboration with a USGS and a USDA scientist whose research expertise in nitrogen transformation in sediments and aquatic environments, along with the use and management of livestock manure in sediments, will further enhance and expand the scope of this project. This project will accelerate research programs of two early career scientists at the University of Nebraska, foster collaborations with an established USGS biogeochemist and USDA-ARS microbiologist, and will train a PhD student in environmental analytical and stable isotope methods. All activities will help to further our understanding of the potential effects of specific contaminant mixtures on nitrogen biotransformation in floating treatment wetland design to treat run-off from urban and agricultural watersheds. Findings from this study will be disseminated through research presentations, high impact journal articles, local seminars, national conferences, technical workshops, a public magazine article, public outreach presentations, and an undergraduate wetlands class.

Animal Health Component

25%

Research Effort Categories

Basic

75%

Applied

25%

Developmental

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
133	0210	2020	100%

Knowledge Area
133 - Pollution Prevention and Mitigation;

Subject Of Investigation
0210 - Water resources;

Field Of Science
2020 - Engineering;

Keywords

contaminants of emerging concern

ecosystem services

nutrients

wetlands

Goals / Objectives
The overall goal is of this project is to improve our understanding of the cumulative effect of increased use and occurrence of nitrification inhibitors together with increasing occurrence of residues of veterinary antibiotics on nitrogen transformation and loading to aquatic environments. The objectives will be supported by experiments that include quantitative measurements of the effect of mixtures of a commonly applied nitrification inhibitor together with environmentally relevant concentrations of selected antibiotics likely to occur in runoff from fields receiving livestock manure or municipal biosolids.Our specific research objectives are:RO1: Quantify the removal potential of trace levels of antibiotics, nitrification inhibitors, and nitrate-N in floating treatment wetlands and aquatic soils receiving contaminated agricultural run-off. RO2: Identify the combined effects of antibiotics and nitrification inhibitors on the nitrogen cycle as well as antibiotic transformation in floating treatment wetlands and aquatic soils using a mesocosm experimental design.RO3: Compare the effect of antibiotics and nitrification inhibitors on removal potential for nitrate-N between aquatic soils in agroecosystems with different histories of nitrification inhibitor and manure application.

Project Methods
The experiments will be conducted in a series of bench top and microcosm experiments. Specific methods to be used include:Chemical Analyses: Analytes, dissolved gases, and sample isotopes, including nitrate-15N, nitrous oxide-15N, N2-15N, brmide, nitrate-N, nitrite-, ammonium-N, total nitrogen, dissolved organic carbon, selected antibiotics, nitrapyrin, and 15N of plant tissues will be analyzed at the University of Nebraska Water Sciences Laboratory (Lincoln, NE). The graduate student supported on this project will be trained in most, if not all, of the analytical methods needed to conduct the research and after training will have full access to the instrumentation.Antibiotics and degradation products will be quantified using solid phase extraction coupled to liquid chromatography tandem mass spectrometry (LC-MS/MS) on a Waters Quattro Micro or Agilent 6410 triple quadrupole mass spectrometry system (Snow et al. 2003; Sallach et al. 2015; Jaimes-Correa et al. 2015; Zhang et al. 2013). Nitrapyrin and its metabolite, 6-chloropyridine-2-carboxylic acid (6-CPA), will be determined in water samples using solid phase extraction derivatization gas chromatography quadrupole mass spectrometry on an Agilent 5973 GC/MS (Woodward et al. 2016). Stable isotope analogues will be used for quantification when available and surrogates are used to monitor analyte recovery in each method (Cassada et al. 1994).An Isoprime Tracegas Pre-concentrator system interfaced to an Isoprime dual inlet IRMS will be used following a modified azide chemical reduction method to measure 15N of dissolved nitrogen species (McIlvin and Altabet 2005). 15N-enriched reaction products will be analyzed either directly using gas chromatography-mass spectrometry on an Agilent 5793 GC/MS with a GS-GasPRO PLOT column, or in diluted samples using isotope ratio mass spectrometry using a GVI Isoprime continuous flow isotope ratio mass spectrometer interfaced to a Eurovector 3028HT Elemental Analyzer. Dissolved organic carbon will be analyzed using EPA Method 415.2 and 9060A on a OI Model 1010 Analytical Organic Carbon Analyzer, while nitrate-N (EPA Method 353.2), ammonium-N (EPA Method 351.2), total Kjeldahl nitrogen (EPA 351.2), total nitrogen (EPA Method 415.1 and 9060A), will be measured using automated colorimetry on a Seal Analytical AQ2 autoanalyzer. An SCP Science Digi-PREP HT digestion block is available for the digestion methods. Bromide will be measured using EPA Method 300.1 on a Dionex ICS-90 ion chromatograph. Descriptions of methods are included in U.S. EPA Methods for Chemical Analysis of Water and Wastewater (US EPA 1983).Hourly NO3-N and DOC concentrations will be measured from the floating treatment wetland mesocosms using a S::CAN Spectro::LyserTM automatic UV-Vis spectrophotometer probe (S::CAN Measuring Systems-Vienna, Austria) connected to a Multiplexor Pumping System (MPS). The MPS will includee: 18 in-line three-way solenoid valves and manifold, a peristaltic pump fitted with a H-bridge for current inversion, 0.32 cm polypropylene tubing, a 4 mm quartz flow-through cuvette (Starnacells® model 46-Q-4), a storage tubing spool, and programmable Arduino microcontroller (Arduino, www.arduino. cc). The spectrophotometer probe, an instrument designed to measure absorbance from 220 nm to 742.5 nm at 2.5 nm intervals, has been successfully used to measure NO3-N and DOC concentrations in the water column in previous wetland studies (Birgand et al., 2016; Etheridge et al., 2014). The spectrometer probe will be connected to the MPS, which allows measurements of NO3-N and DOC from all mesocosms at hourly intervals (Messer et al., In Review). The microcontroller will be programmed to switch on a peristaltic pump and open one dedicated solenoid valve at a time for water quality sampling of each mesocosm. The entire system will be flushed with the new incoming sample 15 seconds prior to each reading. Samples will be sent to the 4 mm quartz cuvette positioned between the measurement window of the spectrometer for analysis, and flushed sample water will be stored in the storage spool above the spectrometer. Once the spectrometer reading is completed, the peristaltic pump will reverse the pumping direction and purge the system by routing water back into the sampled mesocosm. Each sample period will take approximately 3 to 4 minutes from start to finish. The cuvette will be serviced at least every 48 hr, which was imperative to ensure data will not be affected by biological and chemical fouling. During the service the cuvette will be soaked in oxalic acid solution for up to 10 min before being rinsed with deionized water. Lastly, data stored on the spectrometer will offloaded to a field computer during service times and backup on the UNL server.Molecular Analyses: Nucleic acids will be extracted from frozen sediment and soil samples collected during the mesocosm experiments. The abundance of specific microbial genes involved in nitrification (bacterial and archeal amoA) and denitrification (nirK, nirS, and nosZ) will be determined using quantitative PCR and compared to total 16s rRNA genes to determine how treatments affected the abundance of key microbial groups. Microbial community composition will be determined from sediment-extracted DNA and 16S rRNA gene amplification and sequencing using dual indexed, Illumina compatible fusion primers following the protocol described in Kozich et al. (2013).

Progress 05/01/23 to 04/30/24

Outputs
Target Audience:In this reporting period, our target audience was the professional research community focused on emerging contaminants, agricultural engienering and wetland science/engineering Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided research training opportunities for 3 undergraduate students at the University of Kentucky as well as three graduate students. One graduate student in Agricultural and Biosystems Engineering has finished her doctoral degree. The other two graduate students (one at Kentucky in ABE) and one at Nebraska in Civil and Environmental Engineering, will be finishing their degrees this year. How have the results been disseminated to communities of interest?The results have been disseminated through peer reviewed publications, national conferences and local conferences in Kentucky. What do you plan to do during the next reporting period to accomplish the goals?We plan to finish analysis of data for 15N experimetns, and finalize publications from the project.

Impacts
What was accomplished under these goals? In this progress period, weevaluated the combined effects of 3 common-use veterinary pharmaceuticals (VPs) (lincomycin, monensin, sulfadimethoxine) and a nitrification inhibitor (nitrapyrin) on nitrate-N removal efficiency using mesocosm floating treatment wetlands during the growing seasons of 2022 and 2023. Mesocosm trials conducted in 2022 and 2023 were housed at the University of Kentucky North Farm, a covered hoop house facility located in Lexington, Kentucky. Mesocosms were established two years prior to the study. Floating treatment wetland (FTW) mesocosms were 0.64 m (height) by 1.35 m (length) by 0.79 m (width), with an approximate maximum volume capacity of 378.5 L (Figure 1). Pre-cut foam mats were purchased from Beemats (New Smyrna Beach, FL), and Kentucky native species were planted in the mats during July 2021 to allow time to mature. The species utilized in this experiment included a mixture of broadleaf cattail (Typha latifolia), fox sedge (Carex vulpinoidea), common rush (Juncus effusus), and blue flag stem (Iris virginica). Six NO3-N removal trials were assessed under the influence of various agrochemical solutions. Excluding one trial, NO3-N concentrations were observed to be removed to or below detectable levels in nearly all treatments by day 10 of each trial. Significant differences were observed by treatment and by day, with observations indicating implications of VPs on NO3-N removal is primarily limited to the first 48 hours for these systems. In comparison to other FTW NO3-N removal mesocosm experiments, rates were similar for treatments that did not have VPs or nitrapryin administered (i.e. FTW), indicating that our study met standard conditions for constructed wetland ecosystem function These results demonstrate that the impact of VPs on wetland ecosystem services may largely be limited by experimental conditions including time, study media, agrochemical concentration, etc. Further, factors such as exposure time, and potentially bioavailability, as the microbial populations may only have a short time to interact with the chemicals before they are degraded, sorbed, or removed by plant uptake. Findings from this study indicate that the addition of agrochemical mixtures significantly increases NO3-N removal in wetland ecosystems. Additionally, plant, water and soil results observations from this study indicate VP uptake and persistence is prevalent in all three, albeit unequally, with water having the largest recovery pool. Several studies have investigated one or several of these compartments, but to our knowledge, no investigation has been conducted on the fate and transport of constructed wetland mesocosm NO3-N removal when a mixture of VPs and NIs are added over several growing seasons

Publications

Type: Journal Articles Status: Published Year Published: 2024 Citation: Russell, M.3, Messer, T.L., Bartelt-Hunt, S., Snow, D.D., Smith, R.L., Repert, D.A., and Reed, A.P. 2024. Influence of Antibiotics and Nitrification Inhibitors on Treatment Wetland Nitrogen Transformation. Toxics. 12(5): 346. https://doi.org/10.3390/toxics12050346
Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Russell, M. V., Messer, T. L., Bartelt-Hunt, S. L., Snow, D. D. Assessing the Impact of Four Veterinary Antibiotic Species on Treatment Wetland Nutrient Removal at the Mesocosm Scale, Society of Environmental Toxicology and Chemistry poster session, Louisville, KY, November 16, 2023.
Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Russell, M. V., Messer, T. L., Bartelt-Hunt, S. L., Snow, D. D. Assessing the impact of Veterinary Antibiotic species on Treatment Wetland Nutrient Removal at the Mesocosm Scale (No. EGU23-8135). Copernicus Meetings. Vienna, Austria, April 27th, 2023.
Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Russell, M. V., Messer, T. L., Bartelt-Hunt, S. L., Snow, D. D. Assessing the Impact of Four Veterinary Antibiotics on Floating Treatment Wetland Nitrogen Processes, American Ecological Engineering Society conference session, Tampa Bay, FL, June 8, 2023.
Type: Other Status: Published Year Published: 2024 Citation: Russell, M. V., Messer, T. L., Bartelt-Hunt, S. L., Snow, D. D., W. Sanderson, W. Ford. Assessing Nutrient Removal Rates in Floating Treatment Wetlands Following Introduction to Common-use Agrochemical Mixtures. University of Kentucky Department of Biological and Agricultural Engineering Seminar. Lexington, Kentucky, March 1st, 2024.
Type: Conference Papers and Presentations Status: Published Year Published: 2024 Citation: Russell, M. V., Messer, T. L., Bartelt-Hunt, S. L., Snow, D. D., W. Sanderson, W. Ford. Assessing Nutrient Removal Rates in Floating Treatment Wetlands Following Introduction to Common-use Agrochemical Mixtures. Southeast Regional Research Symposium. Lexington, Kentucky, March 25th, 2024.

Progress 05/01/22 to 04/30/23

Outputs
Target Audience:In this reporting period, our target audience was the professional research community focused on emerging contaminants, agricultural engienering and wetland science/engineering Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The project has provided training for two graduate students, one in environmental engineering at the University of Nebraska and one in agricultural engineering at the University of Kentucky. Two undergraduate students in mechanical engineering at the Univresity of Kentucky were also engaged in the research activities. How have the results been disseminated to communities of interest?We have disseminated conference presentations to scientific audiences. Manuscripts are in progress and we anticipate two manuscripts to be submtited this summer. What do you plan to do during the next reporting period to accomplish the goals?We will conduct a final set of mesocosm experiments at the University of Kentucky and a final set of microcosm experiments at the Univresity of Nebraska-Lincoln. We will also complete antibiotics analysis of the summer 2022 microcosms.

Impacts
What was accomplished under these goals? Our specific researchobjectivesare: RO1:Quantify the removal potential of trace levels of antibiotics, nitrification inhibitors, and nitrate-N in floating treatment wetlands and aquatic soils receiving contaminated agricultural run-off. RO2:Identify the combined effects of antibiotics and nitrification inhibitors on the nitrogen cycle as well as antibiotic transformation in floating treatment wetlands and aquatic soils using a mesocosm experimental design. RO3: Compare the effect of antibiotics and nitrification inhibitors on removal potential for nitrate-N between aquatic soils in agroecosystems with different histories of nitrification inhibitor and manure application. Under R01 and RO2, we conducted a second round of mesocosm experiments to evaluate the potential for antibiotics and nitrification inhibitors to impact wetland functioning. These experiments were performed at the University of Kentucky. An additional round of mesocosm experiments with N15 tracers will be completed in summer 2023. At the University of Nebraska-Lincoln, we established more wetland mesocosms to expand our study of microplastic inputs on wetland functioning. Those experiments are ongoing and will be completed in Summer 2023.

Publications

Type: Conference Papers and Presentations Status: Published Year Published: 2023 Citation: Sutton, M.K., Miller, D.N., Messer, T., and Bartelt-Hunt, S.L. Investigating the Influence of Microplastic Morphology and Surface Characteristics on Nitrogen Transformation in Floating Treatment Wetlands. SETAC Europe 33nd Annual Meeting, Dublin, Ireland.

Progress 05/01/21 to 04/30/22

Outputs
Target Audience:During this reporting period, our audience has been the professional research community focused on agricultural practices and environmental quality Changes/Problems:Our only concern is that due to the effects of COVID-19, we lost time for research activities in the summer of 2020. We woudl like to request a one year no cost extension for this project to allow for an additional field growing season, as the wetland experiments can be conducted seasonally. What opportunities for training and professional development has the project provided?The project has provided training opportunities for two PhD students, one in environmental engineering and one in biological systems engineering. The project has also provided training for two undergraduate students in research. How have the results been disseminated to communities of interest?We have made numerous conference presentations and are finalizing two manuscripts to be submitted in Fall 2022. What do you plan to do during the next reporting period to accomplish the goals?We are in the process of conducting an additional set of experiments in wetland mesocosms in summer 2022. We will continue wetland microcosm studies evaluating microplastic fate. We will prepare for a final set of experiments to be conducted in summer 2023.

Impacts
What was accomplished under these goals? Our specific researchobjectivesare: RO1:Quantify the removal potential of trace levels of antibiotics, nitrification inhibitors, and nitrate-N in floating treatment wetlands and aquatic soils receiving contaminated agricultural run-off. RO2:Identify the combined effects of antibiotics and nitrification inhibitors on the nitrogen cycle as well as antibiotic transformation in floating treatment wetlands and aquatic soils using a mesocosm experimental design. RO3: Compare the effect of antibiotics and nitrification inhibitors on removal potential for nitrate-N between aquatic soils in agroecosystems with different histories of nitrification inhibitor and manure application. Under each of these objectives, we analyzed data from a preliminary experiment conducted in the summer of 2021. A subsequent set of experiments has been intiated with aquatic soils in summer 2022 and are ongoing at the University of Kentucky in the lab of co-PD Messer. We were able to expand our contaminants evaluated in this study to include microplastics. Mesocosm experiments were conducted in 2021 and 2022. To evaluate the potential impact microplastics may have on nitrogen transformation in these environments, root samples from an established floating treatment wetland were collected and used to inoculate microcosms to study nitrification and denitrification processes. Microplastic treatments included polyethylene (PE) and polystyrene (PS) microspheres at two diameters (30 µm and 200 µm) and three concentrations (10 mg/L, 100 mg/L, and 1000 mg/L). Nitrification microcosms were observed in aerobic conditions over a 24-hour period and denitrification microcosms flushed with nitrogen gas to encourage anoxic conditions were monitored over a week. Nitrite production was analyzed using a microplate method to calculate a potential nitrification and denitrification rates. For nitrification experiments, only PE 30 µm was found to have a significantly lower normalized rate than root controls. For denitrification experiments, PE 200 µm and PS 30 µm showed a normalized rate significantly higher than the root control and PS 200 µm showed a rate significantly lower. For both nitrification and denitrification experiments, between microplastic treatment, there was a significant difference in the normalized rates between the diameters for both plastic types. Additionally, there was not a significant difference found between the three concentrations (10 mg/L, 100 mg/L, and 1000 mg/L) tested. Further research to observe the impacts on weathering and chemical additives would give a better understanding on how microplastics may interact with microorganisms and influence nitrogen movement in wetlands.

Publications

Type: Theses/Dissertations Status: Published Year Published: 2022 Citation: Influence of Polyethylene and Polystyrene Microplastics on Nitrogen Transformation in Floating Treatment Wetlands
Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Russell, M., Messer, T.L., Bartelt-Hunt, S., Snow, D, Smith, R., Repert, D., Reed, A. Influence of Common Veterinary Antibiotics on Treatment Wetland Ecosystem Services at the Mesocosm Scale. American Society of Agricultural and Biological Engineers 2021 Conference. July 12-16, 2021.
Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Messer, T.L., Bartelt-Hunt, S., Russell, M., Lindgren, J., Sutton, M. Implications of Emerging Contaminants on Treatment Wetlands. American Society of Agricultural and Biological Engineers 2021 Conference. July 12-16, 2021.
Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Russell, M., Messer, T.L., Bartelt-Hunt, S., Snow, D, Smith, R., Repert, D., Reed, A. Influence of Common Veterinary Antibiotics on Treatment Wetland Ecosystem Services at the Mesocosm Scale. American Ecological Engineering Society 2021 Conference. 25-26 May 2021.
Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Sutton, M.K., Miller, D.N., Messer, T., and Bartelt-Hunt, S.L. (2021). Influence of Microplastics on Denitrification in Floating Wetland Environments, American Chemical Society Fall Meeting, Atlanta, GA.
Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Sutton, M.K., Miller, D.N., Messer, T., and Bartelt-Hunt, S.L. (2021). Impact of Polyethylene and Polystyrene Microplastics on Nitrogen Transformation in Floating Treatment Wetlands, American Geophysical Union Fall Meeting, New Orleans, LA.
Type: Conference Papers and Presentations Status: Published Year Published: 2022 Citation: Sutton, M.K., Miller, D.N., Messer, T., and Bartelt-Hunt, S.L. (2022). Influence of Microplastics on Rates of Potential Nitrification and Denitrification in Floating Treatment Wetland Systems, Society of Environmental Toxicology and Chemistry Europe Annual Meeting, Copenhagen, Denmark.

Progress 05/01/20 to 04/30/21

Outputs
Target Audience:During this project period, our target audience has been the professional research community. Changes/Problems:We were able to return to our labs within this reporting period (in June 2021). We did complete a mesocosm study after June, but we missed a portion of the growing season in 2021. Depending on progress in the next reporting period, we may request a no cost extension. One project member, CoPD Tiffany Messer moved from UNL to UKY and reestablished her lab during the summer of 2021. What opportunities for training and professional development has the project provided?The project has provided training for two PhD students, one in environmental engineering at the University of Nebraska-Lincoln and one in agricultural engineering at the University of Nebraska-Lincoln but transferring to the University of Kentucky. Two undergraduate students at the University of Kentucky were involved in this project. How have the results been disseminated to communities of interest?We have made five conference presentations in this reporting period and are working on our first journal publication documenting our results of the microcosm and preliminary mesocosm experiments. What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will conduct more mesocosm studies in support of all three research objectives to evaluate antibiotic and nitrapyrin fate in FTWs. We will track antibiotics in nitrapyrin between soil, water and plants within the wetland mesocosms. We will investigate the additional of soil into the wetland mesocosms. We will continue to investigate microplastic fate in FTW root microcosms.

Impacts
What was accomplished under these goals? Theoverall goalis of this project is to improve our understanding of the cumulative effect of increased use and occurrence of nitrification inhibitors together with increasing occurrence of residues of veterinary antibiotics on nitrogen transformation and loading to aquatic environments. The objectives will be supported by experiments that include quantitative measurements of the effect of mixtures of a commonly applied nitrification inhibitor together with environmentally relevant concentrations of selected antibiotics likely to occur in runoff from fields receiving livestock manure or municipal biosolids. Our specific researchobjectivesare: RO1:Quantify the removal potential of trace levels of antibiotics, nitrification inhibitors, and nitrate-N in floating treatment wetlands and aquatic soils receiving contaminated agricultural run-off. RO2:Identify the combined effects of antibiotics and nitrification inhibitors on the nitrogen cycle as well as antibiotic transformation in floating treatment wetlands and aquatic soils using a mesocosm experimental design. RO3: Compare the effect of antibiotics and nitrification inhibitors on removal potential for nitrate-N between aquatic soils in agroecosystems with different histories of nitrification inhibitor and manure application. In support of RO1 and RO2, we conducted mesocosm experiments at the University of Nebraska-Lincoln after our laboratories opened back up in June 2021. Our mesocosm and microcosm data taken together support our preliminary findings that nitrapyrin and antibiotics have a minimal impact of nitrification but increase denitrification. Our working hypothesis is that the antibiotics and nitrapyrin act as a carbon source for denitrifying bacteria. We are currently investigating whether antibiotics are removed in floating treatment wetlands and whether antibiotics were taken up by the plants in this system. We have also had an opportunity to investigate the fate of microplastics in FTWs and impacts on nitrification and denitrification by FTW plant roots. This expands the types of emerging contaminants evaluated in this study. We continued to collaborate with USGS colleagues to investigate nitrification, denitrification and microbial community structure changes in the saturated soil historically impacted by antibiotics.

Publications

Type: Conference Papers and Presentations Status: Published Year Published: 2020 Citation: Russell, M., Messer, T.L., Bartelt-Hunt, S., Snow, D. Assessing Nutrients Removal Rates in Floating Treatment Wetlands Introduced to Common-Use Agricultural Antibiotics. SETAC. 16-18 November 2020.
Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Russell, M., Messer, T.L., Bartelt-Hunt, S., Snow, D, Smith, R., Repert, D., Reed, A. Influence of Common Veterinary Antibiotics on Treatment Wetland Ecosystem Services at the Mesocosm Scale. American Society of Agricultural and Biological Engineers 2021 Conference. July 12-16, 2021.
Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Messer, T.L., Bartelt-Hunt, S., Russell, M., Lindgren, J., Sutton, M. Implications of Emerging Contaminants on Treatment Wetlands. American Society of Agricultural and Biological Engineers 2021 Conference. July 12-16, 2021.
Type: Conference Papers and Presentations Status: Published Year Published: 2021 Citation: Russell, M., Messer, T.L., Bartelt-Hunt, S., Snow, D, Smith, R., Repert, D., Reed, A. Influence of Common Veterinary Antibiotics on Treatment Wetland Ecosystem Services at the Mesocosm Scale. American Ecological Engineering Society 2021 Conference. 25-26 May 2021.
Type: Other Status: Other Year Published: 2021 Citation: Messer, T.L. Wetland Ecosystem Resiliency: Current and Future Considerations as Influent Water Chemistry Evolves. Distinguished Lecture Series. American Society of Agricultural and Biological Engineers 2021. Virtual Conference. 13 July 2021.

Progress 05/01/19 to 04/30/20

Outputs
Target Audience:During this reporting period, our target audience has been the professional research community. Changes/Problems:Due to the COVID-19 pandemic, we do not currently have access to research labs at UNL. The date we can return to the lab is uncertain and may result in a delay in the planned mesocosm experiments, and completion of chemical analysis of samples collected during the previous quarter. It is too early to know whether this delay will result in any substantial changes to our project timeline. What opportunities for training and professional development has the project provided?The project provided training for a PhD student at the University of Nebraska-Lincoln pursuing a degree in Civil Engineering. Two undergraduate students were recruited to assist with mesocosm development and were provided with research experience by the project. The project provided training for a USGS intern in compiling data and data presentation. How have the results been disseminated to communities of interest?Over the past year of the project we have engaged in extensive data collection and have not yet begun disseminating project results. What do you plan to do during the next reporting period to accomplish the goals?In the next reporting period, we plan to conduct mesocosm experiments to evaluate the removal potential of trace levels of antibiotics, nitrification inhibitors, and nitrate in floating treatment wetlands and aquatic soils. We will also conduct additional experiments to identify mechanisms for the observed microcosm results as well as quantify nitrogen cycling genes and determine microbial community composition. We plan to develop a peer-reviewed publication based on the microcosm results in the next reporting period.

Impacts
What was accomplished under these goals? 1.Major Activities Completed / Experiments Conducted: Mesocosms were built and plants were established for initiation of experiments either during the summer of 2020 (COVID19 dependent) or summer of 2021. Two Undergraduate Creative Activities and Research Experience (UCARE) students, funded by the University of Nebraska-Lincoln, established the wetland mesocosms during the 2019-2020 year. A PhD student was recruited and set to begin in May 2020 A project kickoff meeting was held in Spring 2019 between the USGS and UNL collaborators to initiate the project, and a Summer 2019 field trip to the US MARC facility was made to identify sediment collection sites. Aquatic sediments were collected from the US MARC facility in Clay Center, Nebraska and sent to the USGS lab in Boulder, CO for microcosm experiments. Three sediments with varying degrees of antibiotic exposure were evaluated. Preliminary incubation experiments were performed in August 2019. The objectives of these preliminary experiments were to: (1) modify laboratory methods and procedures to accommodate for the relative nitrogen cycling rates of each site and (2) quantify denitrification & nitrification rate potentials in soils with varying, historical exposure to antibiotic mixtures from agricultural wastewater. A second set of microcosm experiments was performed using sediments collected from the area with the highest historic antibiotic exposure. These soils were collected in December 2019 and shipped to the USGS in Boulder, CO where experiments were performed from December 2019 through January 2020 to simulate in-field effects of antibiotic mixtures and nitrification inhibitors on denitrification and nitrification rate potentials. 2.Data Collected: For the August 2019 preliminary experiments, bulk water and aquatic sediments from each of the three US MARC locations was analyzed. For each sediment type, the anaerobic denitrification potential was evaluated in the presence of a spike antibiotic mixture using an acetylene block assay. An additional anaerobic denitrification experiment was performed with sediment from one of the three locations in the presence of 1 µM sulfamethoxazole. Experiments were also performed for aerobic ammonium loss, aerobic nitrate production and aerobic nitrate + nitrite production. In December 2019, sediments were pre-incubated with either an antibiotic mixture or a nitrification inhibitor for a three-week period before experiments were started. Anaerobic denitrification potential and aerobic nitrification potential were evaluated in the presence of antibiotics and nitrapyrin. Nitrapyrin was added via a spiking solution made in dimethyl sulfoxide (DMSO). 3.Summary Statistics and Discussion of Results: Preliminary data (August 2019) indicated that sediments from the location with moderate antibiotic exposure had reduced denitrification potential relative to the control location (no historic antibiotic exposure). Also, the addition of antibiotics (mixture and SMX only) in lab expts showed a moderate to significant inhibitory effect on the anaerobic consumption of nitrite at the moderate exposure site. There was an increase in the rate of aerobic ammonium loss with increasing history of antibiotic exposure and a higher rate of aerobic nitrate production was observed in the soil with the most extensive history of antibiotic exposure. For the December 2019 microcosm experiments, anaerobic denitrification rates were higher in the presence of spiked antibiotics than in sediments receiving nitrapyrin treatments. Denitrification rates were higher with higher spiked antibiotic concentrations (200 and 1000 ppb) than lower concentrations (0 and 20 ppb). The denitrification rate for sediments receiving 1000 ppb nitrapyrin was greater than sediments receiving 0, 20 and 200 ppb nitrapyrin. DMSO solvent used to deliver nitrapyrin also appears to inhibit denitrification. Results of aerobic nitrification experiments showed no significant difference in nitrification rates between nitrapyrin dosages and that higher antibiotic concentrations stimulated nitrification.

Publications