Stockpiling versus Composting: Effectiveness in Reducing Antibiotic-Resistant Bacteria and Resistance Genes in Beef Cattle Manure.

Manure storage methods can affect the concentration and prevalence of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in cattle manure prior to land application. The objective of this study was to compare stockpiling and composting with respect to their effectiveness in reducing ARB and ARGs in beef cattle manure in a field-scale study. Field experiments were conducted in different seasons with different bulking agents for composting. For both the winter-spring cycle and the summer-fall cycle, ARB concentrations declined below the limit of quantification rapidly in both composting piles and stockpiles; however, ARB prevalence was significantly greater in the composting piles than in the stockpiles. This was likely due to the introduction of ARB from bulking agents. There was no significant change in ARG concentrations between initial and final concentrations for either manure storage treatment during the winter-spring cycle, but a significant reduction of the ARGs erm(B), tet(O), and tet(Q) over time was observed for both the composting pile and stockpile during the summer-fall cycle. Results from this study suggest that (i) bulking agent may be an important source of ARB and ARGs for composting; (ii) during cold months, the heterogeneity of the temperature profile in composting piles could result in poor ARG reduction; and (iii) during warm months, both stockpiling and composting can be effective in reducing ARG abundance. IMPORTANCE Proper treatment of manure is essential to reduce the spread of antibiotic resistance and protect human health. Stockpiling and composting are two manure storage methods which can reduce antibiotic-resistant bacteria and resistance genes, although few field-scale studies have examined the relative efficiency of each method. This study examined the ability of both methods in both winter-spring and summer-fall cycles, while also accounting for heterogeneity within field-scale manure piles. This study determined that bulking agents used in composting could contribute antibiotic-resistant bacteria and resistance genes. Additionally, seasonal variation could hinder the efficacy of composting in colder months due to heterogeneity in temperature within the pile; however, in warmer months, either method of manure storage could be effective in reducing the spread of antibiotic resistance.

Influence of Setback Distance on Antibiotics and Antibiotic Resistance Genes in Runoff and Soil Following the Land Application of Swine Manure Slurry.

The environmental spread of antibiotics and antibiotic resistance genes (ARGs) from the land application of livestock wastes can be a potential public health threat. The objective of this study was to assess the effects of setback distance, which determines how close manure may be applied in relation to surface water, on the transport of antibiotics and ARGs in runoff and soil following land application of swine manure slurry. Rainfall simulation tests were conducted on field plots covered with wheat residues, each of which contained an upslope manure region where slurry was applied and an adjacent downslope setback region that did not receive slurry. Results show that all three antibiotics (chlortetracycline, lincomycin, and tiamulin) and seven out of the ten genes tested (erm(B), erm(C), intI1, tet(O), tet(Q), tet(X), and the 16S rRNA gene) decreased significantly in runoff with increased setback distance. Only blaTEM, chlortetracycline, and tiamulin decreased significantly in surface soil with increased setback distance, while the other analytes did not exhibit statistically significant trends. By using linear regression models with field data, we estimate that a setback distance between 34-67 m may allow manure-borne antibiotics and ARGs in runoff to reach background levels under the experimental conditions tested.

Dehydration of Prions on Environmentally Relevant Surfaces Protects Them from Inactivation by Freezing and Thawing.

Chronic wasting disease (CWD) is an emerging prion disease in North America. Recent identification of CWD in wild cervids from Norway raises the concern of the spread of CWD in Europe. CWD infectivity can enter the environment through live animal excreta and carcasses where it can bind to soil. Well-characterized hamster prion strains and CWD field isolates in unadsorbed or soil-adsorbed forms that were either hydrated or dehydrated were subjected to repeated rounds of freezing and thawing. We found that 500 cycles of repeated freezing and thawing of hydrated samples significantly decreased the abundance of PrPSc and reduced protein misfolding cyclic amplification (PMCA) seeding activity that could be rescued by binding to soil. Importantly, dehydration prior to freezing and thawing treatment largely protected PrPSc from degradation, and the samples maintained PMCA seeding activity. We hypothesize that redistribution of water molecules during the freezing and thawing process alters the stability of PrPSc aggregates. Overall, these results have significant implications for the assessment of prion persistence in the environment.IMPORTANCE Prions excreted into the environment by infected animals, such as elk and deer infected with chronic wasting disease, persist for years and thus facilitate horizontal transmission of the disease. Understanding the fate of prions in the environment is essential to control prion disease transmission. The significance of our study is that it provides information on the possibility of prion degradation and inactivation under natural weathering processes. This information is significant for remediation of prion-contaminated environments and development of prion disease control strategies.

Assessing the Accuracy of Citizen Scientist Reported Measurements for Agrichemical Contaminants.

Citizen science is a research tool capable of addressing major environmental challenges, including contamination of water resources by agrichemicals, such as nutrients and pesticides. The objectives of this study were (1) to identify the proportion of accurate observations by citizen scientists using rapid assessment water quality tools, and (2) to characterize how a user's prior experience with water quality tools was associated with the accuracy of citizen scientists. To achieve these objectives, we conducted group testing with over 136 citizen scientists and compared their results from water quality testing of water samples to results obtained using laboratory analytical methods. Following brief training, we observed that accuracy of reported results varies based on the user's experience level where experienced and expert users shared consistent and reliable measurements. Where erroneous measures were reported, citizen scientists tend to overestimate contaminant concentrations when using colorimetric water quality tools. Additionally, we identified differences in accuracy related to the types of water quality assessment tools used by citizen scientists from each experience group. This study demonstrates the importance of evaluating participant background experience in designing citizen science campaigns.

Stereoselective Degradation of Estradiol and Trenbolone Isomers in Alluvial Sediment.

Stereoisomers of estradiol (E2) or trenbolone (TB) can occur together in the environment receiving human or livestock wastes. However, the effect of their co-occurrence on persistence has not been well elucidated. A sandy and a silt loam sediment were used to establish microcosms with α- and ß-isomers of E2 or TB spiked individually and together. Sediments were sampled periodically and analyzed for E2 and TB isomers and their transformation products using derivatization gas chromatography-mass spectrometry. Results showed that stereoselective degradation was significant for E2 in both sediments and TB in the sandy sediment with ß-isomers decaying more rapidly than α-isomers. In the sandy sediment containing limited natural organic carbon and nutrients, co-occurrence of both isomers of either E2 or TB decreased the dissipation rates. In the silt loam sediment with abundant organic matter and nutrients, the decay rates of both isomers were not changed in the presence of the other isomer. Estrone (E1) and trendione (TD) were detected as primary metabolites of E2 and TB isomers, respectively. The formation and decay profiles of E1 were similar in both sediments with 92-100% of E2 transformed to E1. The TD profiles were different across sediments with ∼100% of TB transformed to TD except in the sandy sediment where 51-60% of 17α-TB was converted to TD. These results indicate that the transformation processes of steroid hormone are stereoselective in sediment and co-occurrence of stereoisomers can prolong steroid persistence and thus pose greater environmental risk.

Bioavailability and Fate of Sediment-Associated Progesterone in Aquatic Systems.

The environmental fate and bioavailability of progesterone, a steroid hormone known to cause endocrine-disrupting effects in aquatic organisms, is of growing concern due to its occurrence in the environment in water and sediment influenced by wastewater treatment plant and paper mill effluents, as well as livestock production. The objective of this study was to evaluate the fate of progesterone in two natural sediments and the corresponding alteration of gene expression in three steroid-responsive genes; vitellogenin, androgen receptor and estrogen receptor alpha. When exposed to progesterone-spiked sand, fathead minnows (Pimephales promelas) exhibited significant reductions in the expression of vitellogenin and androgen receptor expression. In contrast, fish exposed to progesterone associated with the silty loam sediment did not show a biological response at 7 days and only realized a significant reduction in vitellogenin. In both sediments, progesterone degradation resulted in the production of androgens including androsteinedione, testosterone, and androstadienedione, as well as the antiestrogen, testolactone. Differences in compound fate resulted in organism exposure to different suites of metabolites either in water or associated with the sediment. Results from this study suggest that environmental progestagens will lead to defeminization at environmentally relevant concentrations, and that exposure is influenced by sediment properties.

Detection, Occurrence and Fate of Emerging Contaminants in Agricultural Environments.

A total of 59 papers published in 2015 were reviewed ranging from detailed descriptions of analytical methods, to fate and occurrence studies, to ecological effects and sampling techniques for a wide variety of emerging contaminants likely to occur in agricultural environments. New methods and studies on veterinary pharmaceuticals, steroids, antibiotic resistance genes in agricultural environments continue to expand our knowledge base on the occurrence and potential impacts of these compounds. This review is divided into the following sections: Introduction, Analytical Methods, Steroid Hormones, Pharmaceutical Contaminants, Transformation Products, and "Antibiotic Resistance, Drugs, Bugs and Genes".

Impact of Sediment on Agrichemical Fate and Bioavailability to Adult Female Fathead Minnows: A Field Study.

Precipitation induced runoff is an important pathway for agrichemicals to enter surface water systems and expose aquatic organisms to endocrine-disrupting compounds such as pesticides and steroid hormones. The objectives of this study were to investigate the distribution of agrichemicals between dissolved and sediment-bound phases during spring pulses of agrichemicals and to evaluate the role of suspended sediment in agrichemical bioavailability to aquatic organisms. To accomplish these objectives, suspended sediment and water samples were collected every 3 days from a field site along the Elkhorn River, located at the downstream end of a heavily agricultural watershed, and were screened for 21 pesticides and 21 steroids. Adult female fathead minnows (Pimephales promelas) were exposed in field mesocosms to river water containing varying sediment loads. Changes in organism hepatic gene expression of two estrogen-responsive genes, vitellogenin (VTG) and estrogen receptor alpha (ERα), as well as the androgen receptor (AR) were analyzed during periods of both low and high river discharge. Trends in agrichemical concentrations of both the dissolved and sediment phases as a function of time show that, while sediment may act as both a source and a sink for agrichemicals following precipitation events, the overall driver for molecular defeminization in this system is direct exposure to the sediment-associated compounds. This study suggests that endocrine disrupting effects observed in organisms in turbid water could be attributed to direct exposure of contaminated sediment.

Persistence of pharmaceuticals in effluent-dominated surface waters.

Pharmaceutical fate in surface water depends on a combination of physical and chemical processes, but currently, little information is available on cumulative dissipation rates in effluent-dominated receiving waters. In this study, dissipation rates were calculated for pharmaceutical compounds in two streams receiving municipal wastewater effluents using results from passive samplers. Seventeen pharmaceuticals were detected at the two sampling locations, and first-order dissipation rates were found to range between 0.03 and -0.02 h, indicating that some compounds were conserved, while others rapidly dissipated over several kilometers downstream of the source.

Narrow grass hedges reduce tylosin and associated antimicrobial resistance genes in agricultural runoff.

Agricultural runoff from areas receiving livestock manure can potentially contaminate surface water with antimicrobials and antimicrobial resistance genes (ARGs). The objective of this study was to investigate the effectiveness of narrow grass hedges (NGHs) on reducing the transport of antimicrobials and ARGs in runoff after land application of swine manure slurry. Plot-scale rainfall simulation tests were conducted on 0.75 m by 4.0 m plots designed to test three treatment factors: manure amendment (control plots receiving no manure vs. amended plots receiving manure based on 3 times N requirement), NGH (plots with a NGH vs. plots without a NGH), and rainfall events (days 1-3). Runoff generated during three 30-min simulated rainfall events was sampled and analyzed for antimicrobials and ARGs. Manure amendment was responsible for the presence of antimicrobial tylosin ( < 0.0001) and tylosin resistance gene (B) ( < 0.0001) in runoff. Narrow grass hedges proved to be effective in reducing tylosin ( < 0.0001) and (B) ( < 0.0347) in runoff. Manure amendment was responsible for the introduction of tylosin ( < 0.0482) and (B) ( = 0.0128) into the soil; however, it had no significant impact on the abundance of the 16S rRNA gene in soil. Results from this study suggest that NGHs could be a best management practice to control the transport of antimicrobials and ARGs in agricultural runoff.

Detection, Occurrence and Fate of Emerging Contaminants in Agricultural Environments.

A total of 43 papers published in 2014 were reviewed ranging from detailed descriptions of analytical methods, to fate and occurrence studies, to measuring and predicting biological effects for a wide variety of emerging contaminants likely to occur in agricultural environments. New methods and studies on veterinary pharmaceuticals, natural and synthetics steroids, and antibiotic resistance genes in agricultural environments continue to expand our knowledge base on the occurrence and potential impacts of these compounds. This review is divided into the following sections: Introduction, Analytical Methods, Occurrence and Fate, Antibiotic Resistance Genes, and Risk Assessment.

Fate and transport of antimicrobials and antimicrobial resistance genes in soil and runoff following land application of swine manure slurry.

Due to the use of antimicrobials in livestock production, residual antimicrobials and antimicrobial resistance genes (ARGs) could enter the environment following the land application of animal wastes and could further contaminate surface and groundwater. The objective of this study was to determine the effect of various manure land application methods on the fate and transport of antimicrobials and ARGs in soil and runoff following land application of swine manure slurry. Swine manure slurries were obtained from facilities housing pigs that were fed chlortetracyline, tylosin or bacitracin and were land applied via broadcast, incorporation, and injection methods. Three rainfall simulation tests were then performed on amended and control plots. Results show that land application methods had no statistically significant effect on the aqueous concentrations of antimicrobials in runoff. However, among the three application methods tested broadcast resulted in the highest total mass loading of antimicrobials in runoff from the three rainfall simulation tests. The aqueous concentrations of chlortetracyline and tylosin in runoff decreased in consecutive rainfall events, although the trend was only statistically significant for tylosin. For ARGs, broadcast resulted in significantly higher erm genes in runoff than did incorporation and injection methods. In soil, the effects of land application methods on the fate of antimicrobials in top soil were compound specific. No clear trend was observed in the ARG levels in soil, likely because different host cells may respond differently to the soil environments created by various land application methods.

Effect of composting on the fate of steroids in beef cattle manure.

In this study, the fate of steroid hormones in beef cattle manure composting is evaluated. The fate of 16 steroids and metabolites was evaluated in composted manure from beef cattle administered growth promotants and from beef cattle with no steroid hormone implants. The fate of estrogens (primary detected as estrone), androgens, progesterone, and the fusarium metabolite and implant α-zearalanol was monitored in manure compost piles. First-order decay rates were calculated for steroid half-lives in compost and ranged from 8 d for androsterone to 69 d for 4-androstenedione. Other steroid concentration data could not be fit to first-order decay models, which may indicate that microbial processes may result in steroid production or synthesis in composting systems. We demonstrate that composting is an effective strategy to remove steroid hormones from manure. Total steroid hormone removal in composted beef cattle manure ranged from 79 to 87%.

COVID-19 related antiviral drugs are less adsorbable on sediment under alkaline and high cation conditions.

The COVID-19 pandemic resulted in unprecedented usage and elevated environmental concentrations of antiviral drugs. However, very limited studies have reported their sorption characteristics on environmental matrices. This study investigated the sorption of six COVID-19 related antivirals on Taihu Lake sediment with varied aqueous chemistry. Results showed that the sorption isotherms for arbidol (ABD), oseltamivir (OTV), and ritonavir (RTV) were linear, while the Freundlich model was the best-fit for ribavirin (RBV) and the Langmuir model for favipiravir (FPV) and remdesivir (RDV). Their distribution coefficient, Kd, varied between 5.051 L/kg to 248.6 L/kg with the sorption capacities ranked as FPV > RDV > ABD > RTV > OTV > RBV. Alkaline conditions (pH 9) and elevated cation strength (0.05 M to 0.1 M) decreased the sorption capacities of the sediment for these drugs. Thermodynamic analysis revealed that the spontaneous sorption of RDV, ABD, and RTV was between physisorption and chemisorption while FPV, RBV, and OTV were mainly physisorption. Functional groups related to hydrogen bonds, π - π interaction, and surface complexation were implicated in the sorption processes. These findings enhance our understanding about the environmental fate of COVID-19 related antivirals and provide basic data for predicting their distribution and risk in the environment.

Analysis of Wastewater Samples to Explore Community Substance Use in the United States: Pilot Correlative and Machine Learning Study.

BACKGROUND: Substance use disorder and associated deaths have increased in the United States, but methods for detecting and monitoring substance use using rapid and unbiased techniques are lacking. Wastewater-based surveillance is a cost-effective method for monitoring community drug use. However, the examination of the results often focuses on descriptive analysis. OBJECTIVE: The objective of this study was to explore community substance use in the United States by analyzing wastewater samples. Geographic differences and commonalities of substance use were explored. METHODS: Wastewater was sampled across the United States (n=12). Selected drugs with misuse potential, prescriptions, and over-the-counter drugs and their metabolites were tested across geographic locations for 7 days. Methods used included wastewater assessment of substances and metabolites paired with machine learning, specifically discriminant analysis and cluster analysis, to explore similarities and differences in wastewater measures. RESULTS: Geographic variations in the wastewater drug or metabolite levels were found. Results revealed a higher use of methamphetamine (z=-2.27, P=.02) and opioids-to-methadone ratios (oxycodone-to-methadone: z=-1.95, P=.05; hydrocodone-to-methadone: z=-1.95, P=.05) in states west of the Mississippi River compared to the east. Discriminant analysis suggested temazepam and methadone were significant predictors of geographical locations. Precision, sensitivity, specificity, and F1-scores were 0.88, 1, 0.80, and 0.93, respectively. Finally, cluster analysis revealed similarities in substance use among communities. CONCLUSIONS: These findings suggest that wastewater-based surveillance has the potential to become an effective form of surveillance for substance use. Further, advanced analytical techniques may help uncover geographical patterns and detect communities with similar needs for resources to address substance use disorders. Using automated analytics, these advanced surveillance techniques may help communities develop timely, tailored treatment and prevention efforts.

Occurrence, transmission, and zoonotic potential of chronic wasting disease.

Chronic wasting disease (CWD) is a fatal, transmissible prion disease that affects captive and free-ranging deer, elk, and moose. Although the zoonotic potential of CWD is considered low, identification of multiple CWD strains and the potential for agent evolution upon serial passage hinders a definitive conclusion. Surveillance for CWD in free-ranging populations has documented a continual geographic spread of the disease throughout North America. CWD prions are shed from clinically and preclinically affected hosts, and CWD transmission is mediated at least in part by the environment, perhaps by soil. Much remains unknown, including the sites and mechanisms of prion uptake in the naive host. There are no therapeutics or effective eradication measures for CWD-endemic populations. Continued surveillance and research of CWD and its effects on cervid ecosystems is vital for controlling the long-term consequences of this emerging disease.

Replication efficiency of soil-bound prions varies with soil type.

Prion sorption to soil is thought to play an important role in the transmission of scrapie and chronic wasting disease (CWD) via the environment. Sorption of PrP to soil and soil minerals is influenced by the strain and species of PrP(Sc) and by soil characteristics. However, the ability of soil-bound prions to convert PrP(c) to PrP(Sc) under these wide-ranging conditions remains poorly understood. We developed a semiquantitative protein misfolding cyclic amplification (PMCA) protocol to evaluate replication efficiency of soil-bound prions. Binding of the hyper (HY) strain of transmissible mink encephalopathy (TME) (hamster) prions to a silty clay loam soil yielded a greater-than-1-log decrease in PMCA replication efficiency with a corresponding 1.3-log reduction in titer. The increased binding of PrP(Sc) to soil over time corresponded with a decrease in PMCA replication efficiency. The PMCA efficiency of bound prions varied with soil type, where prions bound to clay and organic surfaces exhibited significantly lower replication efficiencies while prions bound to sand exhibited no apparent difference in replication efficiency compared to unbound controls. PMCA results from hamster and CWD agent-infected elk prions yielded similar findings. Given that PrP(Sc) adsorption affinity varies with soil type, the overall balance between prion adsorption affinity and replication efficiency for the dominant soil types of an area may be a significant determinant in the environmental transmission of prion diseases.

Effect of growth promotants on the occurrence of endogenous and synthetic steroid hormones on feedlot soils and in runoff from beef cattle feeding operations.

Supplements and growth promotants containing steroid hormones are routinely administered to beef cattle to improve feeding efficiency, reduce behavioral problems, and enhance production. As a result, beef cattle manure will contain both synthetic steroids as well as a range of endogenous steroids including androgens, estrogens, and progestogens. A two-year controlled study was conducted in which beef cattle were administered steroid hormones via subcutaneous implants and feed additives and the occurrence of 16 endogenous and synthetic steroid hormones and metabolites was evaluated in runoff from beef cattle feedlots and in manure and soil collected from feedlot surfaces. Samples were extracted and analyzed using liquid chromatography tandem mass spectrometryfor metabolites of the synthetic androgen trenbolone acetate, 17α-trenbolone, 17ß-trenbolone, for the nonsteroidal semisynthetic estrogen agonist, α-zearalanol, and the synthetic progesterone melengesterol acetate, as well as a wide range of endogeneous estrogens, androgens, and fusarium metabolites. Synthetic steroids including trenbolone metabolites and melengestrol acetate were detected in fresh manure and in feedlot surface soils from cattle administered synthetic steroids at concentrations up to 55 ± 22 ng/g dry weight (dw) (17α-trenbolone) and 6.5 ± 0.4 ng/g dw (melengesterol acetate). Melengesterol acetate was detected in 6% of runoff samples from feedlots holding cattle administered synthetic steroids at concentrations ranging up to 115 ng/L. The presence of melengesterol acetate in runoff from beef cattle feeding operations has not been previously reported. Synthetic steroids were not detected in manure or runoff from control cattle. A wide range of endogenous hormones were detected in runoff and feedlot surface soils and manure from cattle given synthetic steroids and from control cattle, with no statistically significant differences in concentration. These results indicate that runoff from confined animal production facilities is of environmental and public health concern regardless of the use of growth promotants.

Sensitive detection of chronic wasting disease prions recovered from environmentally relevant surfaces.

Chronic wasting disease (CWD) has been identified in 30 states in the United States, four provinces in Canada, and recently emerged in Scandinavia. The association of CWD prions with environmental materials such as soil, plants, and surfaces may enhance the persistence of CWD prion infectivity in the environment exacerbating disease transmission. Identifying and quantifying CWD prions in the environment is significant for prion monitoring and disease transmission control. A systematic method for CWD prion quantification from associated environmental materials, however, does not exist. In this study, we developed an innovative method for extracting prions from swabs and recovering CWD prions swabbed from different types of surfaces including glass, stainless steel, and wood. We found that samples dried on swabs were unfavorable for prion extraction, with the greatest prion recovery from wet swabs. Using this swabbing technique, the recovery of CWD prions dried to glass or stainless steel was approximately 30% in most cases, whereas that from wood was undetectable by conventional prion immunodetection techniques. Real-time quake-induced conversion (RT-QuIC) analysis of these same samples resulted in an increase of the detection limit of CWD prions from stainless steel by 4 orders of magnitude. More importantly, the RT-QuIC detection of CWD prions recovered from stainless steel surfaces using this method was similar to the original CWD prion load applied to the surface. This combined surface swabbing and RT-QuIC detection method provides an ultrasensitive means for prion detection across many settings and applications.