Subsurface Manure Injection Reduces Surface Transport of Antibiotic Resistance Genes but May Create Antibiotic Resistance Hotspots in Soils.

Compared to surface application, manure subsurface injection reduces surface runoff of nutrients, antibiotic resistant microorganisms, and emerging contaminants. Less is known regarding the impact of both manure application methods on surface transport of antibiotic resistance genes (ARGs) in manure-amended fields. We applied liquid dairy manure to field plots by surface application and subsurface injection and simulated rainfall on the first or seventh day following application. The ARG richness, relative abundance (normalized to 16s rRNA), and ARG profiles in soil and surface runoff were monitored using shotgun metagenomic sequencing. Within 1 day of manure application, compared to unamended soils, soils treated with manure had 32.5-70.5% greater ARG richness and higher relative abundances of sulfonamide (6.5-129%) and tetracycline (752-3766%) resistance genes (p ≤ 0.05). On day 7, soil ARG profiles in the surface-applied plots were similar to, whereas subsurface injection profiles were different from, that of the unamended soils. Forty-six days after manure application, the soil ARG profiles in manure injection slits were 37% more diverse than that of the unamended plots. The abundance of manure-associated ARGs were lower in surface runoff from manure subsurface injected plots and carried a lower resistome risk score in comparison to surface-applied plots. This study demonstrated, for the first time, that although manure subsurface injection reduces ARGs in the runoff, it can create potential long-term hotspots for elevated ARGs within injection slits.

Spatial distribution and temporal change of antibiotics in soils amended with manure using two field application methods.

Compared to surface application, manure subsurface injection significantly reduces transport of manure-associated antibiotics via surface runoff. However, the environmental fate of antibiotics in manure injection slits is unknown. A field investigation was conducted to monitor distribution and dissipation of pirlimycin, tylosin, chlortetracycline, and sulfamerazine in soil following either surface application or subsurface injection of liquid dairy manure. A simulated rainfall was conducted on days 0, 3, and 7 after manure application. Soil samples were collected before, on the day of, and 5, 14, 60, and 180 days after the simulated rainfall. Around an hour after manure application, antibiotic concentrations in injection slits were 4-49 and 4-26 times higher than those outside the slits and in surface application plots, respectively. Antibiotics concentrated in the injection slits for an extended time with limited horizontal and vertical movement, exposing the microbial community inside the slits to an elevated level of antibiotics. Dissipation of antibiotics was the fastest during the first 14 d after manure application before slowing down. There were no significant differences in antibiotic dissipation patterns in soils amended with manure using two application methods. Although the half-lives ranged from 3-11 d for pirlimycin, 3-10 d for sulfamerazine, 5-12 d for tylosin, and 3-21 day for chlortetracycline; pirlimycin, sulfamerazine, and tylosin remained detectable in soil even 180 d after the single manure application, indicating that soils could be a long-term source for antibiotics to the surrounding environment. Overall, in addition to resulting in less surface runoff of antibiotics from the fields, manure subsurface injection can also retain antibiotics in the injection slits and limit their movement overtime. However, more studies are needed to better understand if elevated levels of antibiotics, nutrients, organic matter, and water would result in "hot zones" for antibiotic resistance development in the manure subsurface injected fields.

Method of Dairy Manure Application and Time before Rainfall Affect Antibiotics in Surface Runoff.

Although research has shown that manure soil subsurface injection reduces nutrient input to the aquatic environment, it is less known if it also reduces antibiotic surface runoff from manure-applied fields. Surface runoff of four dairy production antibiotics was monitored comparing (i) surface application and subsurface injection of manure and (ii) time gaps between manure application and a subsequent rain event. Liquid dairy manure spiked with pirlimycin, tylosin, chlortetracycline, and sulfamerazine was applied to 1.5-m × 2-m test plots at an agronomic N rate via surface application and subsurface injection. On the day of application (Day 0), and 3 and 7 d after manure application, a simulated rainfall (70 mm h) was conducted to collect 30 min runoff. Target antibiotics in runoff water and sediment were quantified using ultra-performance liquid chromatography tandem mass spectrometry. Results demonstrated that runoff was a significant route for transporting antibiotics off manure-applied fields, amounting to 0.45 to 2.62% of their initial input with manure. However, compared with manure surface application, subsurface injection reduced sulfamerazine, chlortetracycline, pirlimycin, and tylosin losses in runoff by at least 47, 50, 57, and 88%, respectively. Antibiotic distribution between aqueous and solid phases of runoff was largely determined by water solubility and partition capacity of antibiotics to soil particles. Masses in the aqueous phase were 99 ± 0.5, 94 ± 4, 91 ± 7, and 22 ± 15% of pirlimycin, sulfamerazine, tylosin, and chlortetracycline, respectively. Manure application 3 d or longer before a subsequent rain event reduced antibiotic runoff by 9 to 45 times. Therefore, using subsurface injection and avoiding manure application <3 d before rain would be a recommended manure land management best practice.

Manure Injection Affects the Fate of Pirlimycin in Surface Runoff and Soil.

Antibiotics used in animal agriculture are of increasing environmental concern due to the potential for increased antibiotic resistance after land application of manure. Manure application technology may affect the environmental behavior of these antibiotics. Therefore, rainfall simulations were conducted on plots receiving three manure treatments (surface application, subsurface injection, and no manure control) to determine the fate and transport of pirlimycin, an antibiotic commonly used in dairy production. Rainfall simulations were conducted immediately and 7 d after application of dairy manure spiked with 128 ng g (wet weight) pirlimycin. Soil samples were collected from all plots at two depths (0-5 and 5-20 cm). For injection plots, soil was collected from injection slits and between slits. Pirlimycin concentrations were higher in soil within the injection slits compared with surface application plots at 0 and 7 d. Pirlimycin concentrations in the 0- to 5-cm depth decreased by 30, 55, and 87% in the injection slit, between injection slits, and surface application plots 7 d after application. Pirlimycin concentrations were 106 ng g in sediment and 4.67 ng mL in water from the surface application plots, which were 21 and 32 times that of the injection plots, respectively. After 7 d, pirlimycin levels in runoff sediment and water decreased 80 to 98%. Surface application resulted in six and three times higher pirlimycin concentrations in water and sediment than injection. These results indicate that pirlimycin is most susceptible to loss immediately after manure application. Thus, injection could be considered a best management practice to prevent loss of antibiotics in surface runoff.

Multiple Applications of Sodium Bisulfate to Broiler Litter Affect Ammonia Release and Litter Properties.

Ammonia (NH) emissions from animal manures can cause air and water quality problems. Poultry litter treatment (PLT, sodium bisulfate; Jones-Hamilton Co.) is an acidic amendment that is applied to litter in poultry houses to decrease NH emissions, but currently it can only be applied once before birds are placed in the houses. This project analyzed the effect of multiple PLT applications on litter properties and NH release. Volatility chambers were used to compare multiple, single, and no application of PLT to poultry litter, all with and without fresh manure applications. A field component consisted of two commercial broiler houses: one had a single, preflock PLT application, while the other received PLT reapplications during the flock using an overhead application system. In the volatility chambers, single and reapplied PLT caused greater litter moisture and lower litter pH and , relative to no PLT. After 14 d, NH released from litter treated with reapplied PLT was significantly less than litter with both single and no applications. Furthermore, total N in litter was greatest in litter treated with reapplied PLT, increasing its fertilizer value. In the commercial poultry houses, PLT reapplication led to a temporary decrease in litter pH and , but these effects did not last because of continued bird excretion. Although one preflock PLT application is currently used as a successful strategy to control NH during early flock growth, repeat PLT application using the overhead reapplication system was not successful because of problems with the reapplication system and litter moisture concerns.

Novel manure management technologies in no-till and forage introduction to the special series.

Surface application of manures leaves nitrogen (N) and phosphorus (P) susceptible to being lost in runoff, and N can also be lost to the atmosphere through ammonia (IH3) volatilization. Tillage immediately after surface application of manure moves manure nutrients under the soil surface, where they are less vulnerable to runoff and volatilization loss. Tillage, however, destroys soil structure, can lead to soil erosion, and is incompatible with forage and no-till systems. A variety of technologies are now available to place manure nutrients under the soil surface, but these are not widely used as surface broadcasting is cheap and long established as the standard method for land application of manure. This collection of papers includes agronomic, environmental, and economic assessments of subsurface manure application technologies, many of which clearly show benefits when comparedwith surface broadcasting. However, there remain significant gaps in our current knowledge, some related to the site-specific nature of technological performance, others related to the nascent and incomplete nature of the assessment process. Thus, while we know that we can improve land application of manure and the sustainability of farming systems with alternatives to surface broadcasting, many questions remain concerning which technologies work best for particular soils, manure types, and farming and cropping systems.

Manure application technology in reduced tillage and forage systems: a review.

Managing manure in reduced tillage and forage systems presents challenges, as incorporation by tillage is not compatible. Surface-applied manure that is not quickly incorporated into soil provides inefficient delivery of manure nutrients to crops due to environmental losses through ammonia (NH3) volatilization and nutrient losses in runoff, and serves as a major source of nuisance odors. An array of technologies now exist to facilitate the incorporation of liquid manures into soil with restricted or minor soil disturbance, some of which are new: shallow disk injection; chisel injection; aeration infiltration; pressure injection. Surface banding of manure inforages decreases NH3 emissions relative to surface broadcasting, as the canopy can decrease wind speed over the manure, but greater reductions can be achieved with manure injection. Soilaeration is intended to hasten manure infiltration, but its benefits are not consistent and may be related to factors such as soildrainage characteristics. Work remains to be done on refining its method of use and timing relative to manure application, which may improve its effectiveness. Placing manure under the soil surface efficiency by injection offers much promise to improve N use efficiency through less NH3 volatilization, reduced odors and decreased nutrient losses in runoff, relative to surface application. We identified significant gaps in our knowledge as manyof these technologies are relatively new, and this should help target future research efforts including environmental, agronomic, and economic assessments.

Modifying broiler diets with phytase and vitamin D metabolite (25-OH D(3)): impact on phosphorus in litter, amended soils, and runoff.

Adding phytase and 25- hydroxycholecalciferol (25-OH D(3)) to broiler diets has been shown effective at reducing total P concentrations in broiler litter. This study was conducted to determine the impact of field application of broiler litter from modified diets on P solubility in litter-amended soils and P losses in runoff. Five broiler diets and their resulting litters were evaluated: a high P diet, a low P diet, each of those basal diets with phytase added, and a low P diet with phytase and 25-OH D(3) added. A field study was initiated at two sites with each of the five broiler litters and a commercial P fertilizer (triple superphosphate [TSP]) applied at the same total P rate (150 kg P ha(-1)) and a control where no P was applied. Soil P was monitored over time at two depths (0-5 cm and 0-15 cm) soils were collected in the spring and fall to perform rainfall simulation studies. Broiler litter or TSP application increased soil water-soluble P and Mehlich 3-P concentrations relative to the control, however there were no consistent differences detected between litter treatments. Results from the rainfall simulation experiments indicate that diet modification with phytase or 25-OH D(3) does not increase the potential for P losses in runoff from amended soils relative to traditional diets. Moreover, broiler diet modification to reduce excreted P could be a potentially effective method for reducing watershed scale P surpluses in areas of intensive broiler production, without raising concerns over soluble P losses from litter-amended soils.

Critical evaluation of the implementation of mitigation options for phosphorus from field to catchment scales.

Nutrient regulations have been developed over the past decades to limit anthropogenic inputs of phosphorus (P) to surface waters. All of the regulations were promulgated in response to decreased water quality, which was at least partially associated with agricultural non-point source pollution. Improvements in water quality can take years, so the impacts of these regulations on water quality can not always be seen. Denmark has had nutrient management regulations aimed at achieving mass balance of P for 20 yr, and although great progress has been made, an average surplus of 11 kg P ha(-1) remains. Northern Ireland is also trying to move toward mass balance, but decreases in inorganic P fertilizer use have been undermined by an increase in the use of feed concentrates. In the Chesapeake Bay watershed, which covers several states in the USA, a variety of best management practices are starting to have an effect on P losses from agriculture, but water quality has only improved slightly. Impairment to the supply of drinking water to the City of Tulsa Oklahoma led to a lawsuit that has greatly affected the management of poultry litter in the supplying watershed. This paper discusses the different regulations that have developed in these four regions, evaluates the strategies used to prevent non-point source pollution of P, reports impacts on water quality, and looks for lessons that can be learned as we move forward.

Evaluating long-term nitrogen- versus phosphorus-based nutrient management of poultry litter.

Environmental concerns are driving manure management in many areas from a traditional nitrogen (N) basis toward phosphorus (P)-based nutrient management plans. We investigated how changing nutrient management from an N to a P basis affected crop yields and soil properties in high P soils over a 7-yr period. Three sites were established on farmers' fields, and at each site the same six treatments were applied for 6 or 7 yr. These treatments were (i) no P; (ii) poultry litter applied on an N basis; (iii) inorganic P, equal to the P applied in treatment 2; (iv) poultry litter applied on an estimated annual crop P removal basis; (v) inorganic P, equal to the P applied in treatment iv; and (vi) poultry litter applied once every 2 or 3 yr at a 2- or 3-yr crop removal P rate. All treatments received the same rate of plant-available N. Yields, P balance, soil pH, Mehlich 1 P, and water-soluble P (WSP) were monitored during the experiment. Over the course of the experiment, litter had the beneficial effect of raising soil pH relative to the inorganic treatments. After 7 yr, Mehlich 1 P and WSP were greatest in soils under the N-based treatments, smallest in the no P treatment, and intermediate in the P-based treatments. For example, at the Shenandoah site, Mehlich 1 P decreased by 35 mg kg(-1) under the no P treatment and increased by 36 mg kg(-1) under the inorganic N-based treatment. There were no significant differences between inorganic fertilizer and poultry litter nutrient sources. The results of this study show that soil test P can be decreased in high-P soils over a few years by changing from an N-based to a P-based nutrient management plan or stopping P applications without negatively affecting yields.

Broiler diet modification and litter storage: impacts on phosphorus in litters, soils, and runoff.

Modifying broiler diets to mitigate water quality concerns linked to excess phosphorus (P) in regions of intensive broiler production has recently increased. Our goals were to evaluate the effects of dietary modification, using phytase and reduced non-phytate phosphorus (NPP) supplementation, on P speciation in broiler litters, changes in litter P forms during long-term storage, and subsequent impacts of diets on P in runoff from litter-amended soils. Four diets containing two levels of NPP with and without phytase were fed to broilers in a three-flock floor pen study. After removal of the third flock, litters were stored for 440 d at their initial moisture content (MC; 24%) and at a MC of 40%. Litter P fractions and orthophosphate and phytate P concentrations were determined before and after storage. After storage, litters were incorporated with a sandy and silt loam and simulated rainfall was applied. Phytase and reduced dietary NPP significantly reduced litter total P. Reducing dietary NPP decreased water-extractable inorganic phosphorus (IP) and the addition of dietary phytase reduced NaOH- and HCl-extractable organic P in litter, which correlated well with orthophosphate and phytic acid measured by 31P nuclear magnetic resonance (NMR), respectively. Although dry storage caused little change in P speciation, wet storage increased concentrations of water-soluble IP, which increased reactive P in runoff from litter-amended soils. Therefore, diet modification with phytase and reduced NPP could be effective in reducing P additions on a watershed scale. Moreover, efforts to minimize litter MC during storage may reduce the potential for dissolved P losses in runoff.

Evaluation of phosphorus transport in surface runoff from packed soil boxes.

Evaluation of phosphorus (P) management strategies to protect water quality has largely relied on research using simulated rainfall to generate runoff from either field plots or shallow boxes packed with soil. Runoff from unmanured, grassed field plots (1 m wide x 2 m long, 3-8% slope) and bare soil boxes (0.2 m wide and 1 m long, 3% slope) was compared using rainfall simulation (75 mm h(-1)) standardized by 30-min runoff duration (rainfall averaged 55 mm for field plots and 41 mm for packed boxes). Packed boxes had lower infiltration (1.2 cm) and greater runoff (2.9 cm) and erosion (542 kg ha(-1)) than field plots (3.7 cm infiltration; 1.8 cm runoff; 149 kg ha(-1) erosion), yielding greater total phosphorus (TP) losses in runoff. Despite these differences, regressions of dissolved reactive phosphorus (DRP) in runoff and Mehlich-3 soil P were consistent between field plots and packed boxes reflecting similar buffering by soils and sediments. A second experiment compared manured boxes of 5- and 25-cm depths to determine if variable hydrology based on box depth influenced P transport. Runoff properties did not differ significantly between box depths before or after broadcasting dairy, poultry, or swine manure (100 kg TP ha(-1)). Water-extractable phosphorus (WEP) from manures dominated runoff P, and translocation of manure P into soil was consistent between box types. This study reveals the practical, but limited, comparability of field plot and soil box data, highlighting soil and sediment buffering in unamended soils and manure WEP in amended soils as dominant controls of DRP transport.

Soil testing to predict phosphorus leaching.

Subsurface pathways can play an important role in agricultural phosphorus (P) losses that can decrease surface water quality. This study evaluated agronomic and environmental soil tests for predicting P losses in water leaching from undisturbed soils. Intact soil columns were collected for five soil types that a wide range in soil test P. The columns were leached with deionized water, the leachate analyzed for dissolved reactive phosphorus (DRP), and the soils analyzed for water-soluble phosphorus (WSP), 0.01 M CaCl2 P (CaCl2-P), iron-strip phosphorus (FeO-P), and Mehlich-1 and Mehlich-3 extractable P, Al, and Fe. The Mehlich-3 P saturation ratio (M3-PSR) was calculated as the molar ratio of Mehlich-3 extractable P/[Al + Fe]. Leachate DRP was frequently above concentrations associated with eutrophication. For the relationship between DRP in leachate and all of the soil tests used, a change point was determined, below which leachate DRP increased slowly per unit increase in soil test P, and above which leachate DRP increased rapidly. Environmental soil tests (WSP, CaCl2-P, and FeO-P) were slightly better at predicting leachate DRP than agronomic soil tests (Mehlich-1 P, Mehlich-3 P, and the M3-PSR), although the M3-PSR was as good as the environmental soil tests if two outliers were omitted. Our results support the development of Mehlich-3 P and M3-PSR categories for profitable agriculture and environmental protection; however, to most accurately characterize the risk of P loss from soil to water by leaching, soil P testing must be fully integrated with other site properties and P management practices.