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Ractopamine and Other Growth-Promoting Compounds in Beef Cattle Operations: Fate and Transport in Feedlot Pens and Adjacent Environments

  • J. K. Challis*
    J. K. Challis
    Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, Canada
    *Email: [email protected]
  • S. Sura
    S. Sura
    Agriculture and Agri-Food Canada (AAFC), Morden, Manitoba R6M 1Y5, Canada
    More by S. Sura
  • J. Cantin
    J. Cantin
    Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, Canada
    Agriculture and Agri-Food Canada (AAFC), Lethbridge, Alberta T1J 4B1, Canada
    More by J. Cantin
  • A. W. Curtis
    A. W. Curtis
    Agriculture and Agri-Food Canada (AAFC), Lethbridge, Alberta T1J 4B1, Canada
    More by A. W. Curtis
  • K. M. Shade
    K. M. Shade
    Agriculture and Agri-Food Canada (AAFC), Lethbridge, Alberta T1J 4B1, Canada
    More by K. M. Shade
  • T. A. McAllister
    T. A. McAllister
    Agriculture and Agri-Food Canada (AAFC), Lethbridge, Alberta T1J 4B1, Canada
  • P. D. Jones
    P. D. Jones
    Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, Canada
    More by P. D. Jones
  • J. P. Giesy
    J. P. Giesy
    Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, Canada
    Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, Canada
    Department of Environmental Science, Baylor University, Waco, Texas 76798, United States
    More by J. P. Giesy
  • , and 
  • F. J. Larney
    F. J. Larney
    Agriculture and Agri-Food Canada (AAFC), Lethbridge, Alberta T1J 4B1, Canada
    More by F. J. Larney
Cite this: Environ. Sci. Technol. 2021, 55, 3, 1730–1739
Publication Date (Web):January 15, 2021
https://doi.org/10.1021/acs.est.0c06450

Copyright © 2021 American Chemical Society. This publication is licensed under

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Abstract

The current study represents a comprehensive investigation of the occurrence and fates of trenbolone acetate (TBA) and metabolites 17α-trenbolone (17α-TBOH), 17β-TBOH, and trendione (TBO); melengesterol acetate (MGA); and the less commonly studied β-andrenergic agonist ractopamine (RAC) in two 8 month cattle feeding trials and simulated rainfall runoff experiments. Cattle were administered TBA, MGA, or RAC, and their residues were measured in fresh feces, pen floor material, and simulated rainfall runoff from pen floor surfaces and manure-amended pasture. Concentrations of RAC ranged from 3600 ng g–1, dry weight (dw), in pen floor to 58 000 ng g–1 in fresh feces and were, on average, observed at 3–4 orders of magnitude greater than those of TBA and MGA. RAC persisted in pen floors (manure t1/2 = 18–49 days), and contamination of adjacent sites was observed, likely via transport of windblown particulates. Concentrations in runoff water from pen floors extrapolated to larger-scale commercial feedlots revealed that a single rainfall event could result in mobilization of gram quantities of RAC. This is the first report of RAC occurrence and fate in cattle feedlot environments, and will help understand the risks posed by this chemical and inform appropriate manure-management practices.

This publication is licensed under

CC-BY-NC-ND 4.0.
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  • by licence
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  • nd licence

Introduction

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In North America, growth-promoting compounds including synthetic hormones, antibiotics, and β-adrenergic agonists are administered to beef cattle to improve health and increase feed efficiency. (1) Manure generated from these feedlots not only contains beneficial nutrients and organic matter but also microorganisms, pharmaceuticals, and steroidal hormones that have the potential to adversely affect the health of wildlife or functions of the environment in general. (2−5) Veterinary pharmaceuticals, including steroidal hormones trenbolone acetate, melengestrol acetate, (1,6) β-agonist ractopamine, (7,8) and associated metabolites, are commonly used to promote the growth of cattle in North American feedlots. A key challenge for feedlot managers and policymakers is to manage manure to optimize its economic value while minimizing environmental risks from associated veterinary pharmaceuticals. (9)
Trenbolone acetate (TBA) is a synthetic, androgenic steroid used as an anabolic growth promoter administered via implants at the base of the ear and is excreted primarily as 17α-trenbolone (17α-TBOH) in feces. (1,6) Melengestrol acetate (MGA) is a synthetic progestogen orally administered to heifers as a feed supplement to improve feed conversion, promote growth, and suppress estrus. (1) MGA is excreted primarily in feces and to a lesser extent in the urine. (6) Ractopamine (RAC) is a β-adrenergic agonist used to promote feed efficiency and is administered as a feed additive for up to 42 days prior to slaughter. (7,8,10,11) RAC is eliminated rapidly in cattle and swine, with 95% of the ingested amount (by mass) excreted within 3 days. (11) While RAC is known to be eliminated in both urine (45%) and feces (55%), (11,12) quantitative measurements of metabolites in cattle have focused on urine, where the monoglucuronide conjugate comprises 95% of the compound mass excreted. (13,14) These growth-promoting compounds have been detected in feedlot manure, runoff, catch-basin water, and particulate matter, as well as surrounding surface waters and soils. (2,5−7,9,15−22) However, concerns about their toxicity to aquatic nontarget organisms such as fish have mostly focused on TBA and metabolites 17α- and 17β-TBOH. (23−27) Studies have observed altered sex ratios in zebra fish (Danio rerio) and fathead minnow (Pimephales promelas) skewed toward male fish as a result of near environmentally relevant exposures (10–25 ng L–1) to 17β-TBOH. (23,26) However, to the best of our knowledge, these effects have never been observed under practical production conditions in the field.
Ractopamine is banned or restricted in China, Russia, the European Union, and 157 other countries, while 27 countries, including the United States, Canada, Japan, Brazil, and South Korea, classify it as safe for use in livestock. (10,11) While many countries have cited concerns over data gaps relating to acceptable residues in meat, (11) the greater knowledge gap relates to RAC as a potential environmental contaminant and toxicant in nontarget organisms given its widespread use in beef and swine production in approved countries. (28) RAC is a full β2-receptor agonist and partial β1 agonist. (29) Adverse effects, including agonism of the trace amine-associated receptor, (28) binding to the estrogen receptor (ER)-α, (30) production of vitellogenin, and increased ER-α and -β transcription in medaka (Oryzias latipes), (31) have been observed in a select number of in vitro studies, all at concentrations in the μg L–1 range. One in vivo study of zebra fish revealed apical effects related to exploratory behavior and locomotion at exposure concentrations between 0.85 and 8.5 μg L–1. (32) The same study reported induction of lipid peroxidation and elevated thiol content in the brain of zebra fish at 0.2 μg L–1. FDA registration and safety trials found that RAC is not carcinogenic in chronically exposed rats and mice and that the acceptable daily intake of 1.25 μg kg–1 day–1 is not exceeded based on residues in target tissues of exposed livestock. (33) However, this did not consider residues of the major glucuronide conjugate that can be hydrolyzed in vivo releasing free RAC and thus contribute to total body burdens. (11)
The occurrence of RAC in the environment has been investigated by relatively few studies. groundwater associated with swine and beef cattle facilities in Nebraska found RAC concentrations ranging from 134 to 524 ng L−1 at swine-associated lagoons to less than the limit of quantification at cattle-associated lagoons and a single detection of 54 ng L−1 at one of the swine facility groundwater sites. (19) In comparison, RAC was observed in wastewater from Malaysian cattle feedlots at concentrations ranging from 140 to 500 ng L–1 and from swine farms between 3000 and 30 000 ng L–1. (16) Only two studies have investigated the occurrence of RAC in surface waters, both reporting concentrations <1 ng L–1, downstream of municipal wastewater effluents (34) and across two sampling seasons in an agriculture-dominant watershed. (22) Although mechanisms of transport and pathways of RAC and other veterinary pharmaceuticals and hormones are yet to be fully elucidated, there is evidence that deposition of particulate matter containing veterinary-use chemicals represents an important pathway leading to contamination of environments adjacent to feedlots. (7,15,18,35)
Despite the results outlined above, there was yet to be a comprehensive investigation of these additives during full-scale feeding trials, from commercial beef cattle feedlots, and their potential for transport in surface runoff. In the current study, cattle were fed the same diet in a multiyear effort to understand the relative occurrence of 17α-TBOH, 17β-TBOH, MGA, and RAC in fresh feces and pen floor material at a confined research feedlot. To assess the potential for transport via surface runoff, studies of simulated rainfall were conducted, both within cattle pens and on manure-amended pasture. Groundwater wells located within and around the research feedlot and a catch basin (runoff storage pond) were also sampled to monitor for potential contamination. Companion pen floor and catch-basin samples were also collected from commercial feedlots administering these additives in southern Alberta. This work expands upon the knowledge regarding the presence and fate of these compounds, particularly RAC, in feedlot manure, which is currently insufficient to appropriately evaluate risks posed by these compounds in environments affected by feedlots.

Materials and Methods

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Feeding Trials

Cattle were fed a standard 60% (by mass) corn (Zea mays L.) silage–40% barley (Hordeum vulgare L.) grain diet during the growing period and a 93% (by mass) barley grain–7% corn silage diet during the finishing period (beginning 112 days from the start of the trial, Tables S1 and S2) at the Agriculture and Agri-Food Canada (AAFC) Research Centre research feedlot (Lethbridge, AB, Canada) in 2017–2018 and 2018–2019. Cattle were fed for 259 days in 2017–2018 and 273 days in 2018–2019. Cattle were penned in groups of 10 with four pens per treatment (n = 4) and a total of six treatments (240 cattle total), organized in a randomized block design. Treatments were (1) control heifers (no growth promoters), (2) trenbolone acetate + estradiol-implanted heifers (TBA), (3) heifers continuously fed with melengestrol acetate (MGA), (4) control steers (no growth promoters), (5) TBA-implanted steers, and (6) steers implanted with TBA and fed ractopamine hydrochloride for the last 42 days prior to slaughter (TBA+RAC). Cattle received three TBA implants (100 mg + 100 mg + 200 mg) spaced approximately 80 days apart over the course of the feeding trials. MGA was fed over the complete duration of each feeding trial at a rate of 0.4 mg heifer–1 day–1 (104–109 mg total per heifer), with respect for a mandated 24 h withdrawal period prior to slaughter. RAC-treated cattle were fed a total of 14.5 g over 42 days. Complete details of the implant and feed regimens are given in the Supporting Information.
Twenty samples of fresh feces (warm fecal pat) were sampled from each pen on 3 consecutive days, 2 weeks after each of the three TBA implants were administered. This ensured that the peak excretion of TBA in feces (36) was captured (Tables S1 and S2). Twenty samples of pen floor material (mix of manure/urine + cereal straw bedding) were collected within each pen once per month on nearly the same date each month. Each set of 20 samples from a given pen was composited into a single sample for processing and analysis. In addition to this routine schedule, in 2017–2018, further sampling was conducted in TBA+RAC pens during RAC treatment (10 sampling points in a 23 day period) and after cattle had vacated the pens (5, 7, 9, 14, 21, and 36 days post-trial). Samples were stored at −20 °C until they were freeze-dried and ground.

Commercial Feedlots

For comparison to large-scale industry practice, manure samples were also collected from four commercial feedlots at 5–6 sampling points over a 2 year period (2016–2018). Two feedlots used conventional production practices (i.e., TBA implants, MGA to heifers, RAC to steers and heifers) with implants and feed additives dosed at the same concentrations employed in the research feeding trials, and two used natural production practices with no additives administered to cattle. At each feedlot, manure samples (20) were randomly collected within 10 pens, with samples composited by pen. All samples were subject to residue analysis as described below. Samples from the two natural commercial feedlots are not discussed further, since residues of target analytes were not detected.

Simulated Runoff Experiments

To characterize concentrations of TBA and metabolites, MGA, and RAC in surface runoff from pen floors and manure-amended pasture, simulated rainfall runoff experiments were conducted in the summer of 2019 with a portable Guelph Rainfall Simulator II (Figure S1). (37) These experiments simulated the mobility of these chemicals from pen floors to catch basins and from manure-amended soils to surface water during rainfall events. Simulated rainfall water was applied at 126.5 mm h–1, mimicking a 1 in 100 year rainfall event in Lethbridge, following protocols established in similar runoff experiments conducted by our group. (3,38) The commercial feedlot catch basins in Alberta are designed to hold 1 day of a 1 in 30 year rain event (https://www.agriculture.alberta.ca/app19/loadcatchbasin). Therefore, a 1 in 100 year event would not only guarantee runoff accumulation for our experiments over reasonable time frames (<1 h) but also simulate extreme rain events that may push feedlot contaminants to surface waters beyond the “closed feedlot environment”. In total, 15 consecutive 1 L samples were collected for each runoff simulation. Simulations were conducted in triplicate for each treatment (TBA × 3 pens, MGA × 3 pens, RAC × 3 pens) directly in the feedlot pens and on manure-amended pasture for a total of 18 runoff experiments. Transport of these chemicals can be influenced by the moisture content of the manure prior to a rainfall event; therefore, runoff experiments were conducted at least 2 days after the last recorded rainfall event in the region. The total duration of rainfall application was 21 ± 3.5 min across all 18 experiments, depending on surface conditions of each plot, with an average of 2.1 ± 1.6 min to the start of runoff collection. Rainfall simulations on pasture were conducted within 1 h of amending the surface soil with manure taken directly from the feedlot pens and applied at a rate of 6 kg m–2 (wet weight, ww), consistent with practices for irrigated land (60 Mg ha–1, recommended maximum rate). (39) Complete experimental and sampling details can be found in the Supporting Information.

Sampling of Groundwater and Catch Basins

Sixteen groundwater wells, located in interior alleyways and around the perimeter of the AAFC research feedlot (Figure S2), were sampled nine times at monthly intervals in 2018–2019. Details of groundwater wells and sampling can be found in the Supporting Information. Water samples were also taken to assess the movement of growth-promoting chemicals from the research feedlot into the catch basin. The feedlot site drained from south to north toward the catch basin with a 0.35% slope (Figure S2). (40) Samples could only be collected opportunistically when heavy precipitation led to runoff water in the catch basin, an event that occurred only once in October 2019. To complement this sampling event, water samples were also collected from catch basins at three commercial conventional feedlots in October 2019. These samples were highly turbid and required centrifugation prior to solid-phase extraction (SPE) (details in the Supporting Information).

Extraction of Residues

Fresh fecal and pen floor samples were freeze-dried and ground to <2 mm. All samples were shipped on ice to the University of Saskatchewan Toxicology Centre (Saskatoon, SK, Canada) for extraction and analysis. While efficiencies of extraction from all matrices were corrected for by isotope dilution (methanolic standards spiked in all samples at 50 ng), in addition, absolute recovery experiments in water and fecal matrices conducted without isotope dilution were ≥91 ± 8% (n = 3) for all analytes in water samples and ≥86 ± 12% (n = 3) for all analytes in fecal samples except for TBO (48 ± 4%, n = 3). All solid samples (dry weight, dw) were extracted in true replicates of four, corresponding to the four pens per treatment used in the feeding trials. Extraction of solid and aqueous samples by accelerated solvent extraction and SPE are detailed in the Supporting Information.

Instrumental Analysis

Analysis was conducted using a Vanquish UHPLC and Q-Exactive HF Quadrupole-Orbitrap mass spectrometer (Thermo-Fisher, Mississauga, ON). LC separation was achieved with a Kinetex 1.7 μm Biphenyl LC column (100 × 2.1 mm2) (Phenomenex, Torrance, CA) by gradient elution with water and acetonitrile, both containing 0.1% formic acid at a flow rate of 0.35 mL min–1 and a column temperature of 40 °C. Samples were ionized by positive mode heated electrospray ionization (HESI) using a full MS/parallel reaction monitoring method at 120 000/15 000 resolution. Complete details of the chromatographic method, retention times, source parameters, inclusion list ions, and collision energies are provided in the Supporting Information. Method detection limits ranged from 0.05 to 0.8 ng mL–1 (Table S6).

Data Analyses

All data acquisition and processing were conducted using Xcalibur v. 4.2 and TraceFinder v. 4.1 (Thermo-Fisher), respectively. Prism v. 5.01 (GraphPad Software, La Jolla, CA) was used for graphing and statistical analysis. Estimates of variabilities are presented as standard deviations (σ; SDs) of the mean. Based on results of the Shapiro–Wilk normality test (p-value > 0.05), with the exception of RAC fresh fecal samples from 2017–2018 (p = 0.019), all of the other samples were sufficiently described by a normal distribution to allow for the use of parametric statistics. Complete details of statistical analyses can be found in the Supporting Information.

Results and Discussion

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Research Feedlot

Occurrence of TBA and MGA in Feeding Trials

Concentrations of TBA, 17α-TBOH, 17β-TBOH, TBO, and MGA were less than the limits of detection (0.029–0.10 ng g–1, Table S6) in control treatments for all samples collected over both feeding trials. Also, in all samples, TBA and its metabolite, TBO, occurred at concentrations less than their respective limits of detection. The parent compound TBA is fully hydrolyzed to the 17β-TBOH metabolite, which is then oxidized to TBO and in turn reduced to the major metabolite 17α-TBOH (≈95%). (6) Detection of TBO in comparable studies investigating the occurrence of TBA metabolites in implanted cattle varies. A mass ratio of 17α-TBOH/17β-TBOH/TBO of approximately 94:4:2 has been reported, (6) while other studies did not detect TBO, reporting ratios of 98:2:0 (2) and 87:13:0. (17) Averaged over 2 years, both fresh fecal and pen floor samples exhibited a 17α-TBOH/17β-TBOH/TBO ratio ranging from 93:7:0 to 95:5:0. The lack of detection of TBO might relate to the relatively poor recovery of TBO (48%), which is consistent with a previous study using similar methods. (2)
Samples of fresh feces generally contained greater concentrations of 17α-TBOH (average 41 ± 30 ng g–1) and 17β-TBOH (average 3 ± 2 ng g–1) than did samples of pen floor (Figure 1). Since pen floor samples consisted of feces, urine, and bedding and greater concentrations of metabolites have been observed in feces compared to urine, (41) elevated levels in fresh fecal samples were expected. In addition, over time, microbial activity on the pen floor or environmental conditions (e.g., desiccation, UV-light) could further reduce concentrations of these residues and their metabolites. (42−44) However, this systematic difference in concentrations between the two types of samples was somewhat variable for each compound, a result attributed to the heterogeneous nature of the matrices and differing distribution of metabolites between urine and feces. Concentrations of MGA were fairly consistent among sampling events and between years (Figure 1), and there was less distinction between fresh fecal and pen floor samples compared to TBA treatments, likely because MGA was continuously administered in the feed as opposed to slow-release implants used for TBA.

Figure 1

Figure 1. Concentrations of trenbolone acetate metabolites 17α-trenbolone (17α-TBOH) and 17β-TBOH and melengesterol acetate (MGA) during the 2017–2018 (left panel) and 2018–2019 (right panel) feeding trials in fresh fecal samples (Fecal) and pen floor samples (Floor). Data points represent mean ± SD from replicate treatment pens: 12× TBA floor (heifer/steer combined), 36× TBA fresh (heifer/steer, consecutive sampling days combined), and 4× MGA. Floor concentrations represent a single sampling event, while fresh concentrations are a mean of 3 consecutive days of sampling. TBA and TBO were less than the LODs in all samples. The 2017–2018 feeding trial began on October 31, 2017, and TBA implants (denoted by red lines) were administered on November 1 (day 1, 100 mg of TBA), January 23 (day 83, 100 mg of TBA), and April 17 (day 167, 200 mg of TBA). The 2018–2019 feeding trial began on December 11, 2018, and TBA implants were administered on December 12 (day 1, 100 mg of TBA), March 5 (day 84, 100 mg of TBA), and May 28 (day 168, 200 mg of TBA). MGA was fed continuously throughout each of the trials.

Most commercial implants are designed to constantly release TBA over an estimated in vivo lifetime of 100 days, (36,41) which is why implants were administered at ≈80 day intervals in the current study. This resulted in a fairly consistent concentration of 17α-TBOH and 17β-TBOH in fresh fecal and pen floor samples throughout the study (Figure 1 and Tables S7–S10,S13–S16). Overall, concentrations differed maximally by 2–3-fold for 17α-TBOH and 17β-TBOH in fresh samples across time points in 2017–2018, which is likely due to natural variation in feeding trials of this type.
Comparisons with concentrations in samples from commercial feedlots, measured in the current study, provide confidence in the realistic feeding trial conditions and sampling protocols used. Commercial concentrations of 17α-TBOH ranged from 1.2 to 44 ng g–1 and MGA from 0.50 to 15 ng g–1, which agree well with measured feeding trial concentrations: 41 ± 30 ng g–1 17α-TBOH, 3 ± 2 ng g–1 17β-TBOH, and 18 ± 8 ng g–1 MGA (means over both feeding trials). A complete description of the commercial feedlot data can be found in the Supporting Information.
Concentrations of metabolites of TBA in a 40 m3 stockpile of manure from 12 Holstein-Friesian heifers implanted with TBA over an ≈80 day period ranged from 5 to 75 ng g–1 17α-TBOH and from 0.5 to 5 ng g–1 17β-TBOH. (6) Additionally, 0.5–4.5 ng g–1 17α-TBOH and 0.04–0.2 ng g–1 17β-TBOH in liquid manure and 2–19 ng g–1 MGA in feces were reported. (6) In another study conducted on composite surface soil samples from a commercial feedlot in Nevada, only 17α-TBOH at 4–6 ng g–1 (dw) was detected. (45) During a 113 day study of metabolite excretion from TBA-implanted cattle, concentrations in manure ranged from 64 ng g–1 17α-TBOH at peak, dissipating to 10 ng g–1 (dw) over the course of the exposure. (2) In the same study, 17β-TBOH measured 1.0–3.9 ng g–1 and TBO was only detected twice at 1.3 and 2.5 ng g–1. In pen floor samples from cattle 28 days post-TBA implantation, mean concentrations were 21 and 3.1 ng g–1 (dw) for 17α-TBOH and 17β-TBOH, respectively, and TBO was not detected. (17) Concentrations in our pen floor materials (Figure 1 and Tables S7 and S18) were typically elevated but nevertheless comparable to those observed in previous studies.

Occurrence of RAC in Feeding Trials

RAC was not detected in any samples until RAC was administered in the final 42 days of the 2017–2018 feeding trial. Between the start (June 5) and the end (July 17) of RAC administration, concentrations in fresh feces ranged from 27 000 to 58 000 ng g–1 (Figure 2). The only pen floor sampling during the 42 day period in 2017–2018 occurred 13 days after the start of RAC administration and measured 3600 ± 700 ng g–1 (n = 4). The RAC concentrations in fresh feces were 3–4 orders of magnitude greater than concentrations of 17α-TBOH, 17β-TBOH, and MGA measured in this study. This result can be attributed to the more concentrated feed (100 g kg–1 or 30 mg kg–1 of the total diet) used for administering RAC. For comparison, the concentration of MGA in feed was ≈200 mg kg–1.

Figure 2

Figure 2. Concentrations of ractopamine (RAC) during 2017–2018 (top) and 2018–2019 (bottom) feeding trials in fresh fecal samples (Fecal) and pen floor samples (Floor). Data points represent mean ± SD from 4× RAC treatment pens. Fecal concentrations represent single sampling events in 2017–2018 and the mean of 3 consecutive days of sampling in 2018–2019. Floor concentrations are single sampling events in both feeding trials. RAC treatment began on June 5, 2018 (day 216, not shown on the plot) and lasted 42 days until the end of the feeding trial on July 17, 2018 (day 258, red line). Prior to the start of treatment in the 2017–2018 feeding trial, RAC was not detected in any samples (top plot begins on day 231). In 2018–2019, sampling was not conducted during RAC treatment (days 230–274), hence the narrower y-axis scale range. The concentrations measured in 2018–2019 reflect the residual RAC remaining from the 2017–2018 trial. Insets are the first-order kinetic time series plots of the floor concentrations. Linear regression provided the first-order rate constant and manure half-life of RAC in 2017–2018 (summer) and 2018–2019 (winter). Dashed lines represent 95% confidence intervals of the linear regression.

Over the 42 days, RAC was detected in manure and feces at concentrations as great as 96 ng g–1 in >80% of TBA, MGA, and control samples, which suggested movement and contamination from the highly concentrated TBA+RAC-treated pens to the non-RAC pens. However, these concentrations were <1% of those present in RAC pens. Similar cross-contamination from TBA- and MGA-treated pens into non-TBA and -MGA pens was not observed. Contamination of environments adjacent to feedlots via windblown particulates is a well-known phenomenon and has been observed for RAC and other natural and synthetic hormones. (7,15,18,35) Concentrations observed in the non-RAC-treated pens (mean = 13 ng g–1, range = 1–96 ng g–1) are within the range of residues detected on wildflowers within 1 km of feedlots in Texas (40–380 ng g–1) (15) and in wetland sediments in close proximity to a feedlot (5.2 ng g–1). (7) However, this is the first study to report concentrations of RAC in fresh fecal and pen floor samples of RAC-treated cattle, making comparisons to the source concentrations in the TBA+RAC pens difficult. Our conventional commercial feedlot data agree well with the pen floor concentrations from our feeding trials, with mean concentrations between 3000 and 4500 ng g–1 RAC (Tables S19 and S20). Additionally, although RAC is only administered in the final ≈40 days prior to slaughter, at large commercial feedlots, administration of RAC is almost constant as cattle rotate through the RAC finishing stage, which suggests the potential continuous release of this contaminant. This is confirmed by the fact that RAC was detected in 100% of the samples taken from the two conventional commercial feedlots during spring, summer, fall, and winter months between 2016 and 2018 (Tables S19 and S20).

Fate and Dissipation of TBA and MGA during Feeding Trials

Dissipation of the target analytes in material from pen floors is best observed in the latter portion of the 2017–2018 feeding trial (after ≈200 days) when extensive sampling was conducted in the TBA+RAC pens. Concentrations of 17α-TBOH and 17β-TBOH dissipated quickly during the summer months (June and July) following the third and final TBA implant. Where sampling was conducted postfeeding in TBA+RAC treatments (Figure 1), 17α-TBOH concentrations were ≤1 ng g–1 in manure samples 10 days post-trial and below detectable limits 22 days post-trial. This finding suggests a benefit to leaving manure in pens for a period after cattle vacate to facilitate dissipation of hormone residues prior to land application. Concentrations of 17β-TBOH were also less than limits of quantification in all samples taken after May in both the 2017–2018 and 2018–2019 feeding trials. While extended sampling, post-trial, was not conducted for MGA, noticeable dissipation in MGA occurred in June and July of both feeding trials, with concentrations dropping below 5 ng g–1, as compared to concentrations from November to May of 15 ± 5 ng g–1. The observed dissipation of TBA metabolites is consistent with observations during previous studies. During batch soil microcosm experiments conducted with clay loam and sandy soils under aerobic conditions, half-lives ranged from 3 to 12 h for 17α-TBOH and 17β-TBOH. (43) Half-lives of all three TBA metabolites as a function of temperature have been reported to range from 4 to 50 h, depending on the compound and the incubation temperature (5–35 °C). (42) As such, we expect dissipation of these chemicals to be greater during summer than winter, consistent with the results reported here (Figure 1).

Fate and Dissipation of RAC during Feeding Trials

Following sampling of pen floor materials, 13 days after the start of RAC treatment (3600 ± 700 ng g–1) in 2017–2018, the five remaining sampling events occurred to track dissipation after cattle were removed from pens and RAC administration ended. Postfeeding trial dissipation of RAC in pen floor samples was observed (Figure 2), but significant amounts remained (681 ± 450 ng g–1, n = 4) up to 37 days after RAC administration ended. Assuming that the concentration in pen floor materials on day 13 of RAC treatment remained relatively constant until administration ended, dissipation rates can be estimated via first-order kinetics (Figure 2, inset). Over the 37 day post-RAC sampling period (6, 8, 10, 22, and 37 days), RAC degraded with a half-life of ≈18 days over July and August of 2018 (0.04 day–1, r2 = 0.77; Table 1).
Table 1. Ractopamine (RAC) First-Order Manure Degradation Rates Measured In Situ during the 2017–2018 and 2018–2019 Feeding Trials (Figure 2) and RAC In Situ Distribution Coefficients (log Kd, L kg–1) Estimated from Sediment (Cs) and Water (Cw) Concentrations in Commercial Feedlot Catch Basinsa
feeding trialk (day–1)t1/2 (days)r2
2017–2018 (July–August)0.039 ± 0.01118 ± 50.769
2018–2019 (January–April)0.014 ± 0.00249 ± 60.958
feedlotCs (ng g–1)Cw (ng L–1)log Kd
commercial 123440001.79
commercial 2128127 0001.67
commercial 3150621 0001.85
a

The date range provided for each feeding trial indicates the months over which the first-order kinetic data apply to (i.e., summer and winter half-lives, t1/2). Catch-basin water and suspended sediments were taken from three conventional commercial feedlots in October 2019.

Residues of RAC from the 2017–2018 treated pens remained at the start of the 2018–2019 feeding trial, measuring 42 ng g–1 maximally with a mean of 15 ng g–1, and dissipated slowly over the course of the feeding trial to less than the limit of detection by May 2018 (Figure 2). This second set of RAC dissipation data provides a first-order kinetic time series (Figure 2, inset) and an estimated manure degradation rate of ≈49 days from January to April of 2019 (Table 1). These differing degradation rates in manure represent a seasonal effect between 2017–2018 (summer) and 2018–2019 (winter), as rates of degradation in soils have been shown to be a strong function of temperature. (42,44) These are the first reported estimates of a half-life of RAC in feedlot pens.
A period of 126 days separated the final sampling point of the 2017–2018 trial (37 days post-trial on August 23, 2018) and the initial sampling of the 2018–2019 trial (December 26, 2018). Given that pens are cleaned (i.e., all floor material removed) prior to the start of a new feeding trial, this result suggests that the earthen-clay black base layer of the pens may harbor RAC residues for extended periods. Additionally, in contrast to observations during administration of RAC, concentrations at the start of the 2018–2019 trial were greatest in pen floor samples as opposed to feces, which further supports the contention that the source of the RAC was from previous use (i.e., 2017–2018 trial) as opposed to fresh feces from the cattle present for the 2018–2019 feeding trial. Lastly, it has been noted that anaerobic environments (42,43) of deeper soil layers in feedlot pen floors can act as potential reservoirs of veterinary chemicals. (17)
Assuming a constant half-life of 49 (winter) and 18 (summer) days over the 126 day period separating the last and first sampling point of the two feeding trials, between ≈120 and 5 ng g–1 RAC would remain, respectively, in the pens at the start of the new feeding trial on December 26, 2018, which agrees well with observed concentrations (range = 6.0–78.9 ng g–1). These results suggest that manures containing RAC residues can serve as sources of this chemical with the potential to enter surrounding environments via runoff, windblown particulates, or when manure is land-applied. Various composting practices are often employed prior to field application and have proved effective at removing certain veterinary pharmaceuticals present in manure. (46−48) However, efficacy varies for different chemicals, and specific composting studies are yet to be published for RAC. The dissipation rates observed here (Table 1) in pen floors suggest that manure composting should be employed as the best management practice to reduce RAC residues prior to land application. Ongoing composting of manure and soil dissipation studies by our group will provide important information regarding these best management practices of RAC residues in manure.

Surface Runoff

Simulated Rainfall Experiments

Concentrations of residues remained relatively constant over the entire duration of rainfall simulations (15 sequential L), which suggests that there was no significant depletion of any of the target chemicals in the source pen floor material and that compounds desorbed or leached from the manure at a constant rate over the duration of the simulated rainfall, consistent with observations elsewhere for similar compounds. (38) With the exception of RAC (discussed below), this was also evidenced by the fact that concentrations measured in the pen floor material pre- and postrainfall simulations remained relatively constant. However, another explanation for the constant concentrations may be related to the leaching of chemicals from suspended solids collected in the 1 L samples. This could potentially drive concentrations in some of the initial samples prior to the establishment of mass transfer on the runoff surface. MGA was not detected in the pasture runoff experiment (Figure S3). The absence of MGA in runoff from pasture could be the result of specific physiochemical interactions, including sorption on soil surface organic matter. (49) Pasture can attenuate certain chemicals due to interactions with living or dead vegetation and soil organic matter fractions, resulting in reduced mobility and thus transport of MGA in the runoff water samples. (5,38)
Mean concentrations over the 15 L pen floor runoff experiments were 76 ± 60 ng L–1 17α-TBOH, 6 ± 3 ng L–1 17β-TBOH, 24 ± 17 ng L–1 MGA, and 6300 ± 5300 ng L–1 RAC. In pasture runoff, sample concentrations measured 51 ± 30 ng L–1 17α-TBOH, 12 ± 6 ng L–1 17β-TBOH, <LOD MGA, and 3100 ± 2100 ng L–1 RAC. While pen floor and pasture concentrations were not statistically different for any of the target chemicals, in general, the pasture samples appeared to have slightly reduced concentrations compared to the pen floor, likely for the same reasons discussed above for MGA. Total masses of chemicals exported in surface runoff over ≈15 L ranged from 60 ng 17β-TBOH to 63 000 ng RAC in the pen floor and from 116 ng 17β-TBOH to 31 000 ng RAC in pasture (Figure S3). Extrapolating these masses from a 1 × 1 m2 simulation area to full pen areas (273 m2) at the AAFC research feedlot, a rainfall event of this magnitude has the potential to mobilize up to 17.2 mg of RAC per pen or 63 μg m–2, assuming a similar stocking density (10 cattle pen–1) as used here. Considering that the largest commercial feedlot in Alberta has a 75 000 head capacity, it is possible that a heavy rainfall event could leach gram quantities of RAC from a single feedlot. It is important to note that these experiments captured only surface runoff. Infiltration into deeper soil layers in feedlot pens has been observed during rain events (17) and leaching experiments on manure-amended land. (5) This could result in unaccounted compound fractions in the current study, thereby underestimating the total estimated loads mobilized during simulated rainfall events.
The fraction of compound captured in these runoff events was estimated for RAC to better understand the extent of mobilization occurring from manure. This was done for the RAC pasture runoff experiments as a defined amount of manure (6 kg or 2.5 kg dw based on 58% water content (38)) was applied to the 1 m2 plots, allowing for an accurate mass inventory pre- and postrunoff. Using the average measured RAC concentrations in the applied manure samples, prerunoff (435 ± 240 ng g–1 or 1.1 × 106 ng RAC per 1 m2 plot), the total rainfall volume (36 L), and the experimental Kd values (Table 1), the fraction of RAC expected in all runoff water (retained and captured) was estimated to be 19%. This is consistent with the measured mass lost in manure samples postrunoff, which ranged from 17 to 25% (compared to initial manure concentrations prerunoff) in replicate runoff experiments. Based on calculated volumetric runoff coefficients, which provide an estimate of the fraction of incoming water (36 L) that leaves the surface as runoff versus the amount retained, (50) 43 ± 9% of total rainfall was captured in the 15 consecutive 1 L runoff water samples. Therefore, of the 19% total RAC mass, only 8% is expected in the collected runoff samples. This, compared to the measured mass of RAC captured in 15 L from the manure-amended pasture, was 30 900 ng or 2.8% of the total mass present in the 1 m2 plot, agreeing well with the estimated 8%. A part of the uncertainty in this calculation is likely related to the assumption of an equilibrium Kd value, which is likely not applicable to the initial stages of these runoff scenarios, as the manure is saturating with rainwater and mass transfer is slow.
It should be noted that monthly groundwater samples taken from the AAFC research feedlot during the 2018–2019 feeding trial (data not shown) revealed that the target chemicals present in the feces, manure, and runoff samples described previously were not detected in groundwater over the course of sampling. This is likely a result of the hard-packed black interface layer that forms under the manure pack on the pen floors as a result of physical compaction by cattle that has been suggested to be responsible for limiting leaching and infiltration to the groundwater below feedlots. (51)
The concentrations in other simulated runoff experiments conducted in cattle feedlot pens were similar to ours, with median (range) concentrations of 34 (1–390) ng L–1 17α-TBOH and 16 (5–26) ng L–1 17β-TBOH. (17) However, 17α-TBOH, 17β-TBOH, or MGA were not detected in runoff samples from edge-of-field weirs where manure from beef cattle receiving TBA and MGA had been applied. (4) Conversely, in a 17 month study, maximum concentrations from different tile-drained fields receiving animal wastewater from lagoons ranged from 9.7 to 22.7 ng L–1 17α-TBOH (1.0–5.7% detection rate) and 4.1 to 162 ng L–1 17β-TBOH (0.4–3.5% detection rate). (21,52) Based on observations in the current study and those of past studies, it remains that concentrations of 17α-TBOH, 17β-TBOH, and MGA in runoff where these compounds are known to be present (i.e., feedlot waste effluents) occur at low ng L–1 concentrations to below limits of detection. While 17β-TBOH is known to be a potent androgenic toxicant at these concentrations, (23,26) detection frequencies are so low (21,52) that the risk of exposure appears to be minimal in most feedlot-associated environments, especially considering the dilution as runoff reaches a body of water. (2) This assertion is supported by the fact that these compounds were also not detected in commercial feedlot catch-basin samples, as detailed below, despite ongoing TBA and MGA treatments at these feedlots. Concentrations of RAC were up to 1000-fold greater in runoff samples compared to 17α-TBOH, 17β-TBOH, and MGA, which suggested that the RAC concentrations mobilized in feedlot runoff pose a greater potential for contamination of surrounding environments.

Catch-Basin Samples

Concentrations of all target chemicals in the AAFC research feedlot catch basin were less than the limit of detection, except for RAC, which was measured at 140 ng L–1. The pens had been cleaned at the time of sampling (October 2019, between feeding trials), which meant that the major source material of the target chemicals (pen floor material) was not present. This explained the relatively low RAC concentration in water in this catch basin compared to catch basins from commercial feedlots, which had concentrations of 4000–27 000 ng L–1 in water and 234–1506 ng g–1 in sediment (Table 1). Other target compounds were not found in commercial feedlot catch-basin samples. Estimated in situKd values determined from the ratio of solid-to-water phase concentrations of each water sample were in good agreement across the three catch-basin sites, varying maximally by <0.2 log units (1.67–1.85). These values were also in good agreement with log Kd values for RAC in 10 mL batch sorption experiments, ranging from 1.58 to 1.68 in natural topsoil and from 1.53 to 1.64 in natural subsoil. (53) The study reported sorption dominated by hydrophobic interactions with soil organic matter and relevant interactions with charged complexes on clay particles. (53) Given that the pKa of RAC (≈9.4) (53) overlaps the typical pH range of manures from our feeding trials (8.5–9.5), (47) binding to charged complexes is likely to be a relevant mechanism. Despite RAC’s relatively high water solubility (31 g L–1) and moderate polarity (log KOW 2.4), these results suggest that RAC persists in both the solid and liquid fractions of manure, presenting multiple potential pathways into adjacent feedlot environments, consistent with our data from both the feeding trials and runoff experiments.

RAC Mass Balance

A mass balance was conducted to better inform the fate and overall fraction of RAC accounted for in our sampling and analyses. Extensive mass balance inventories in feedlot pen environments are reported elsewhere for both TBA (2,6,17) and MGA (6) and thus are not the focus of this exercise. The daily mass intake of feed (0.003% RAC wt/wt) by the TBA+RAC steers during the course of this study was 11.5 ± 0.2 kg day–1, (54) which equates to a RAC intake of 0.00035 kg day–1 per head. Reported fecal excretion rates range from 1.8 kg (dw) day–1 AU–1 (animal unit) for juvenile steers (2) and heifers weighing 379 ± 31 kg (55) to 27–40 kg (ww) day–1 or 3.1–4.6 kg (dw) day–1, assuming a fecal moisture percentage of 88.5%. (41,56) The shrunk body weights of the TBA+RAC steers throughout the finishing period of this study were 425 ± 14 kg (initial) and 717 ± 8 kg (final), (54) the latter weight being more reflective of the cattle during the final 42 day RAC feeding period prior to slaughter. Thus, a fecal excretion rate on the higher end of the reported range (i.e., 3.1−4.6 kg day–1) may be most appropriate for this RAC mass balance. Using the minimum (1.8 kg day−1), median (3.1 kg day−1), and maximum (4.6 kg day−1) reported fecal excretion rates results in predicted RAC manure concentrations of 106 000, 60 100, and 41 400 ng g–1, respectively, assuming 55% excretion in feces. (11) Fresh samples represent only recently excreted fecal pats and thus attenuation is expected to be negligible, especially given that the observed dissipation rates for RAC in this study are in the order of weeks (18–49 days, Table 1). Compared to the average fresh fecal concentrations observed over the 2017–2018 RAC sampling period (37 000 ± 8800 ng g–1), these measured values account for 35% (minimum excretion), 61% (median), and 89% (maximum) of total RAC expected in cattle feces.
Floor samples represent a more uncertain matrix to account for due to dilution with bedding and mixing in the pen floor, potential attenuation over time, and the contribution of urine to the sample. While 45% of RAC is excreted in the urine, (11) an estimated 95% of this is the monoglucuronide conjugate. (13,14) While RAC metabolites were not targeted in this work, retrospective mining of the full-scan high-resolution (120 000) MS data did not reveal characteristic m/z ions (14) of the glucuronide metabolite in any samples, likely due to poor recoveries during extraction (57,58) (details in the Supporting Information). Additionally, the ratio of urine/feces excretion in steers on a typical high-energy diet is 1:2.75, further suggesting that feces likely represent the major contributor to floor RAC concentrations. (41) Given the reasonable mass balance reflected in the fresh fecal samples described above, RAC floor concentrations measuring approximately 10-fold lower (3600 ng g–1) likely reflects the attenuation occurring in aged manure and subsequent mixing by the cattle. While we do have first-order attenuation rate constants for RAC (Table 1), given that floor manure samples were not taken frequently during RAC feeding, it is very difficult to estimate how “aged” a given floor sample was and thus the extent of attenuation that had occurred prior to the time of sampling.

Environmental Significance

This work presents the first comprehensive characterization of the occurrence and fate of RAC in cattle feedlots and adjacent environments. RAC was found at concentrations in fresh fecal and pen floor samples that were 3–4 orders of magnitude greater than those of the synthetic hormones TBA and MGA studied here. The body of knowledge regarding the occurrence and fate of TBA and MGA, here and elsewhere, suggests limited exposure risk to nontarget aquatic organisms given the small concentrations and infrequent detections in most animal manure-impacted environments. However, scenarios of low dilution in close proximity to high-density livestock operations remain a concern that warrants study on a case-by-case basis. The observed concentrations of RAC in fecal and floor samples, surface runoff from rainfall simulations, and catch basins of commercial feedlots suggest that this compound may pose specific challenges when it comes to management practices for manure intended for agricultural use and protection of edge-of-field aquatic environments. Concentrations of RAC in catch-basin water samples (4000–27 000 ng L–1) exceeded levels causing behavioral effects in zebra fish (≥850 ng L–1) (32) by 5–32-fold. While the lack of detection of all chemicals, including RAC, in the groundwater collected in 2018 and 2019 suggests that these chemicals have limited downward mobility, further research is needed to elucidate the presence of RAC and its major glucuronide metabolite in both soil and aquatic environments adjacent to beef cattle feedlots, especially given the elevated concentrations of RAC observed in manures.
Post-trial sampling demonstrated that floor material left in the pens after cattle were removed results in dissipation of 17α-TBOH to below LODs after >10 days while significant RAC concentrations remained after 37 days post-trial (681 ng g–1). From a manure-management perspective, leaving pens for approximately 2 weeks post-feeding can reduce residues of 17α-TBOH, 17β-TBOH, and MGA to below detection and potentially reduce or eliminate the requirement for manure composting prior to land application. Given the elevated concentrations of RAC observed post-trial, further research should be directed toward understanding its dissipation and fate following land-application of fresh manure, and during manure storage (stockpiling) or processing (composting), and informing optimal manure-management practices, work that is ongoing in our group.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.est.0c06450.

  • Feeding trial diets and sampling dates; simulated runoff and groundwater sampling; methodological extraction and analysis protocols including analytical detection limits; and raw tabular concentration data for feeding trials and commercial feedlot samples (PDF)

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Author Information

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  • Corresponding Author
  • Authors
    • S. Sura - Agriculture and Agri-Food Canada (AAFC), Morden, Manitoba R6M 1Y5, Canada
    • J. Cantin - Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, CanadaAgriculture and Agri-Food Canada (AAFC), Lethbridge, Alberta T1J 4B1, Canada
    • A. W. Curtis - Agriculture and Agri-Food Canada (AAFC), Lethbridge, Alberta T1J 4B1, Canada
    • K. M. Shade - Agriculture and Agri-Food Canada (AAFC), Lethbridge, Alberta T1J 4B1, Canada
    • T. A. McAllister - Agriculture and Agri-Food Canada (AAFC), Lethbridge, Alberta T1J 4B1, Canada
    • P. D. Jones - Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, Canada
    • J. P. Giesy - Toxicology Centre, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, CanadaDepartment of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5C9, CanadaDepartment of Environmental Science, Baylor University, Waco, Texas 76798, United States
    • F. J. Larney - Agriculture and Agri-Food Canada (AAFC), Lethbridge, Alberta T1J 4B1, Canada
  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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The authors thank the three anonymous reviewers for taking their time to review the manuscript and providing helpful comments that contributed to its improvement. This work was funded through the Beef Cattle Research Council. The authors also thank the commercial feedlots that provided access for this work. J.K.C. was supported by the Banting Postdoctoral Fellowship. J.P.G. was supported by the Canada Research Chairs program of the Natural Science and Engineering Research Council of Canada.

References

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  • Abstract

    Figure 1

    Figure 1. Concentrations of trenbolone acetate metabolites 17α-trenbolone (17α-TBOH) and 17β-TBOH and melengesterol acetate (MGA) during the 2017–2018 (left panel) and 2018–2019 (right panel) feeding trials in fresh fecal samples (Fecal) and pen floor samples (Floor). Data points represent mean ± SD from replicate treatment pens: 12× TBA floor (heifer/steer combined), 36× TBA fresh (heifer/steer, consecutive sampling days combined), and 4× MGA. Floor concentrations represent a single sampling event, while fresh concentrations are a mean of 3 consecutive days of sampling. TBA and TBO were less than the LODs in all samples. The 2017–2018 feeding trial began on October 31, 2017, and TBA implants (denoted by red lines) were administered on November 1 (day 1, 100 mg of TBA), January 23 (day 83, 100 mg of TBA), and April 17 (day 167, 200 mg of TBA). The 2018–2019 feeding trial began on December 11, 2018, and TBA implants were administered on December 12 (day 1, 100 mg of TBA), March 5 (day 84, 100 mg of TBA), and May 28 (day 168, 200 mg of TBA). MGA was fed continuously throughout each of the trials.

    Figure 2

    Figure 2. Concentrations of ractopamine (RAC) during 2017–2018 (top) and 2018–2019 (bottom) feeding trials in fresh fecal samples (Fecal) and pen floor samples (Floor). Data points represent mean ± SD from 4× RAC treatment pens. Fecal concentrations represent single sampling events in 2017–2018 and the mean of 3 consecutive days of sampling in 2018–2019. Floor concentrations are single sampling events in both feeding trials. RAC treatment began on June 5, 2018 (day 216, not shown on the plot) and lasted 42 days until the end of the feeding trial on July 17, 2018 (day 258, red line). Prior to the start of treatment in the 2017–2018 feeding trial, RAC was not detected in any samples (top plot begins on day 231). In 2018–2019, sampling was not conducted during RAC treatment (days 230–274), hence the narrower y-axis scale range. The concentrations measured in 2018–2019 reflect the residual RAC remaining from the 2017–2018 trial. Insets are the first-order kinetic time series plots of the floor concentrations. Linear regression provided the first-order rate constant and manure half-life of RAC in 2017–2018 (summer) and 2018–2019 (winter). Dashed lines represent 95% confidence intervals of the linear regression.

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    • Feeding trial diets and sampling dates; simulated runoff and groundwater sampling; methodological extraction and analysis protocols including analytical detection limits; and raw tabular concentration data for feeding trials and commercial feedlot samples (PDF)


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