Evaluating the effect of an anticoccidioidal drug on the nitrogen cycle in agricultural soils

Evaluating the effect of an anticoccidioidal drug on the nitrogen cycle in agricultural soils

Summary

The objective of this dissertation research is to fully assess the impact of Narasin on nitrogen dynamics at a field site near Milford, Delaware. Narasin is a USDA approved anticoccidiodal agent administered to poultry. Agricultural soils are exposed to Narsin when poultry litter is applied as a nitrogen fertilizer. Narasin has a relatively short half-life in soil, but may persist at concentrations in the pg·kg^-1 to ng·kg^-1 range. Because sustainable fertilizer practices are based on known parameters of soil nitrogen cycle variation, microbial inhibition or delayed activity caused by antibiotics may undermine the ability of modeling tools to make strong fertilizer management recommendations, leading to reduced fertilizer use efficiency and increased losses of pollutant N species, including N₂O and NO₃^–.

In the preceding 12 months, four full sets of incubation experiments have been completed. Inorganic nutrient data has been quantified for the majority of the samples collected and headspace samples have been submitted to UC Davis for isotopic analysis of _N2 and quantification of N₂O. One data set has been returned. Samples and data are pending for isotopic analysis of NO₃-N and NH₄-N that will be for quantification of soil nitrification and denitrification rates.

Objectives/Performance Targets

OBJECTIVE 1

 The first objective, quantifying the impact of Narasin on soil nitrogen transformation rates, was to be achieved by conducting a comprehensive series of soil incubation tests designed to simultaneously quantify the rate of mineralization, nitrification, denitrification, N₂O emissions, and accumulation of NO₃^–. The approach is fully described in the project proposal. As of December 2014, this objective has nearly been met. All planned incubation experiments are complete. Mineral N content (NO₃^– and NH₄⁺) has been quantified for the majority of the soil extracts collected. Two complete sets of isotopic N₂ data and one complete set of N₂O emissions data have already been received. The remaining samples have been shipped to UC Davis and analysis is pending. The isotopic signature of mineral N samples is needed to complete each data set and requires that the liquid samples be diffused over a period of about 3 weeks. Presently, 1/3 of the samples have undergone this diffusion process and the solid samples have been shipped to UC Davis for analysis. The remaining diffusions are proceeding at a rate of 60 samples/3 weeks and will be complete by the end of February 2015.

OBJECTIVE 2

 The second objective is to develop and test a dose-response model for the rate of mineralization, nitrification, denitrification, N₂O emission, and potential NO₃^– leaching as a function of soil moisture and sulfadiazine concentration. Achieving this objective is dependent upon the successful completion of Objective 1 and therefore there are no accomplishments to report at present.

Accomplishments/Milestones

Quality control data required to validate the planned methodology to collect isotopic samples for this project was received in January 2014. The first set of incubations subsequently began in February 2014 in which 75 g of the soils collected in August 2013 were treated with ¹⁵N-enriched nutrient substrates and 0, 1, 10, 100, or 1000 ng/kg Narasin. Specifics of the methodologies are described in the original proposal. Total water content was adjusted to 40% water-filled pore space (WFPS). Per the planned methodology, headspace samples were collected from each 6 incubation chambers daily for a total of 3 days and the soils subsequently extracted with 2M KCl. Headspace samples were submitted for N₂ isotope analysis and N₂O quantification. The concentration of NH₄⁺ and NO₃^– in the soil extracts was determined by colorimetric methods and the extracts were diffused to produce solid samples for mineral N isotope analysis. Mineral nitrogen data from this experiment showed no discernible pattern of behavior related to the antibiotic dose and the standard deviation obtained from 6 replicate samples averaged in excess of 30%, making it difficult to draw any substantial conclusions. The poor quality of these results led to further literature review where it was found that re-wetting of dry soils can result in a sudden release of mineral nitrogen over a period of 2-3 days that is not associated with biological activity. Based on this information, the diffusion of soil extracts was halted to avoid costly sample analysis that was unlikely to yield the desired information regarding biologically mediated mineralization, nitrification, and denitrification. The methods were subsequently modified to allow a 4-day “pre-incubation” period before soils were dosed with nutrient solution and antibiotics. Since the original proposal allowed for isotopic analysis of 4 sets of incubation experiments and a portion of these funds were expended on the initial “discovery” experiment, the decision was made to limit the final project to 3 incubations in which the final volume of water (following nutrient and antibiotic amendments) was 40%, 60%, or 80% WFPS and to increase the number of antibiotic dosages tested from 4 to 7. Mineral nutrient and N₂O flux data are being collected for all doses tested whereas the isotopic data (¹⁵N-N₂, ¹⁵N-NH₄⁺, ¹⁵N-NO₃^–) remain limited to the original dosages (1, 10, 100, and 1000 ng·kg^-1_soil). Presently, all of the mineral N data has been obtained for soils incubated at 40% and 60% WFPS. Remaining mineral N analysis is expected to be complete by the end of January 2015. Gas samples for N₂O and ¹⁵N-N₂ data have already been submitted for analysis and the soil extract diffusions for ¹⁵N-NH₄⁺ and ¹⁵N-NO₃^– data required to calculate mineralization, nitrification, and denitrification rate are ongoing. Approximately 1/3 of these incubations are presently complete. An additional incubator shaker is being sought to help expedite this process.

Under the experimental conditions described above mineralization, nitrification, and denitrification will proceed concurrently, thus it is difficult to draw any specific conclusions about the impact of trace Narasin exposure the rate of individual processes until the mineral N isotope data has been received. However, some broad conclusions may be drawn from the total mineral N concentration in soil extracts and from associated N₂O flux data. For instance, at 40% WFPS, soils treated with the highest antibiotic doses (>100 ng·kg^-1_soil) were enriched with NH₄⁺ relative to the initial value (C/C_initial > 1) for the duration of the experiment, which suggests that nitrogen mineralization was stimulated by the presence of the antibiotic at these concentrations (Figure 1). In general, the residual NO₃^– concentration was less than that of the control (no antibiotics added) for all soils treated with antibiotics, but the lowest concentrations of NO₃^– were associated with mid-range antibiotic dosages (10-100 ng·kg^-1_soil) (Figure 2). Whether this is due to an accelerated denitrification rate or an inhibited nitrification rate will become clear when the isotope data becomes available. Also notable in this experiment is the N₂O flux rate shows a distinct dose-response pattern (Figure 3). After one day of incubation, the average N2O flux is approximately 0.1 ppm·day^-1 for all antibiotic treatments and the control. On the second day, this flux rate is maintained only by those soils treated with 10-100 ng·kg^-1_soil Narasin whereas the control and other doses yielded nearly double the flux on this second day. By Day 3, a linear dose-response trend was evident, with N₂O flux ranging from 1 ppm·day^-1 (Control) to approximately 0.4 ppm·day^-1 (1000 ng·kg^-1_soil Narasin). Over the full incubation period, these data show an increase in net N₂O flux as a result of exposure to Narasin. Until the isotope data has been applied, the variations in N₂O flux cannot be definitively attributed to nitrification or denitrification, but the dose-response pattern is most similar to that of NO₃^– and suggests that the increase in N₂O flux is a result of accelerated denitrification. Similar patterns of nutrient N were observed from soils incubated at 60% WFPS though the magnitude of NH₄⁺ accumulation and NO₃^– reduction were less significant.

• Figure 1. Concentration of extractable ammonium in soils treated with 0-1000 ng/kg Narasin over a three day incubation period.
• Figure 2. Concentration of extractable nitrate in soils treated with 0-1000 ng/kg Narasin over a three day incubation period.
• Figure 3. Daily nitrous oxide flux in soils treated with 0-1000 ng/kg Narasin over a three day incubation period.

Impacts and Contributions/Outcomes

At the current stage of this project, it is clear that soils exposed to trace levels of the anticoccidiodal drug Narasin exhibit dose-dependent shifts in nitrogen cycling rates. The exact nature of this shift will be elucidated pending receipt of enriched ¹⁵N isotope data. Despite the mechanistic unknowns that remain, a preliminary assessment of the gross mineral N and N₂O data does have some implications to agricultural sustainability. For instance, under both 40% and 60% WFPS, where coupled nitrification-denitrification tends to predominate, extractable NO₃^– is significantly reduced over a 3-day period by exposure to Narasin at doses ranging from 0.1 to 1 μg·kg^-1_soil. In cases where NO₃^– is the preferred N-source for plant nutrition, this may lead to N-deficiencies and reduced crop yields if fertilizer applications are not adjusted to compensate for this shift. Furthermore, there appears to be an increase in N₂O flux associated with reduced levels of NO₃^–. If the nitrogen fertilization rate were increase to compensate for NO₃^– losses, it may subsequently contribute to even higher N₂O flux, which poses an environmental risk in terms of its greenhouse gas and ozone reduction potential. A comprehensive assessment of this risk will be detailed in the final report associated with this project.

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