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.

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.

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.

Resistome and mobilome in surface runoff from manured soil as affected by setback distance.

Land application of livestock manure introduces antibiotic resistance genes (ARGs) and mobile genetic elements (MGEs) into the soil environment. The objectives of this study were to examine the changes of resistome and mobilome in runoff and soil as a function of setback distance, i.e., the distance between manured soil and surface water, and to quantify the contributions of manure and background soil to the ARGs and MGEs in surface runoff. The resistome and mobilome in runoff and soil from a field-scale plot study were characterized using a high throughput quantitative polymerase chain reaction (HT-qPCR) array. It was estimated that a setback distance of ~40 m is required to reduce the total abundance of ARGs and MGEs in runoff from amended plots to that in control runoff. The resistome and mobilome of the soil in the setback region was not affected by manure-borne ARGs and MGEs. SourceTracker analyses revealed that background soil gradually became the predominant source of the ARGs and MGEs in runoff as setback distance increased. The results demonstrate how manure-borne ARGs and MGEs dissipated in agricultural runoff with increasing setback distance and had limited impacts on the resistome and mobilome of soil within the setback region.

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.

Modeling the vertical transport of antibiotic resistance genes in agricultural soils following manure application.

Antibiotic resistance genes (ARGs) may be introduced to agricultural soil through the land application of cattle manure. During a rainfall event, manure-borne ARGs may infiltrate into subsurface soil and leach into groundwater. The objective of this study was to characterize and model the vertical transport of manure-borne ARGs through soil following the land application of beef cattle manure on soil surface. In this study, soil column experiments were conducted to evaluate the influence of manure application on subsurface transport of four ARGs: erm(C), erm(F), tet(O) and tet(Q). An attachment-detachment model with the decay of ARGs in the soil was used to simulate the breakthrough of ARGs in leachates from the control column (without manure) and treatment (with manure) soil columns. Results showed that the first-order attachment coefficient (ka) was five to six orders of magnitude higher in the treatment column than in the control column. Conversely, the first-order detachment and decay coefficients (kd and µs) were not significantly changed due to manure application. These findings suggest that in areas where manure is land-applied, some manure-borne bacteria-associated ARGs will be attached to the soil, instead of leaching to groundwater in near terms.

Antibiotic resistance genes in swine manure slurry as affected by pit additives and facility disinfectants.

Manure storage facilities are critical control points to reduce antibiotic resistance genes (ARGs) in swine manure slurry before the slurry is land applied. However, little is known about how exogenous chemicals entering the manure storage facilities may affect the fate of ARGs. The objective of this study was to analyze the impact of six commonly used pit additives and four facility disinfectants on the concentration of ARGs in swine manure slurry. Bench scale reactors, each containing approximately 50 L of liquid swine manure, were dosed with additives or disinfectants and were sampled for 40 days. Seven antibiotic resistance genes along with the intI1 gene and the 16S rRNA gene were monitored. Out of the six additives tested, Sludge Away significantly reduced the time-averaged absolute abundance of erm(C), erm(F), tet(Q), and the 16S rRNA gene as compared to the no additive control. Out of the four disinfectants tested, Tek-Trol significantly reduced the time-averaged absolute abundance of erm(B), erm(C), erm(F), intI1, tet(Q), and tet(X) than did the no-disinfectant control. According to Spearman's rank correlation, three genes erm(F), tet(Q), and tet(X) showed a strong to perfectly positive correlation and the two genes erm(B) and tet(O) showed a moderate to strong correlation in both the additive and disinfectant tests. Overall, the disinfectants were more effective in controlling the absolute abundance of ARGs than were the pit additives.

Climate change impacts the subsurface transport of atrazine and estrone originating from agricultural production activities.

Climate change will impact soil properties such as soil moisture, organic carbon and temperature and changes in these properties will influence the sorption, biodegradation and leaching of trace organic contaminants to groundwater. In this study, we conducted a modeling case study to evaluate atrazine and estrone transport in the subsurface under current and future climate conditions at a field site in central Nebraska. According to the modeling results, in the future, enhanced evapotranspiration and increased average air temperature may cause drier soil conditions, which consequently reduces the biodegradation of atrazine and estrone in the water phase. On the other hand, greater transpiration rates lead to greater root solute uptake which may decrease the concentration of atrazine and estrone in the soil profile. Another consequence of future climate is that the infiltration and leaching rates for both atrazine and estrone may be lower under future climate scenarios. Reduced infiltration of trace organic compounds may indicate that lower trace organic concentrations in groundwater may occur under future climate scenarios.

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.

Predicting Escherichia coli loads in cascading dams with machine learning: An integration of hydrometeorology, animal density and grazing pattern.

Accurate prediction of Escherichia coli contamination in surface waters is challenging due to considerable uncertainty in the physical, chemical and biological variables that control E. coli occurrence and sources in surface waters. This study proposes a novel approach by integrating hydro-climatic variables as well as animal density and grazing pattern in the feature selection modeling phase to increase E. coli prediction accuracy for two cascading dams at the US Meat Animal Research Center (USMARC), Nebraska. Predictive models were developed using regression techniques and an artificial neural network (ANN). Two adaptive neuro-fuzzy inference system (ANFIS) structures including subtractive clustering and fuzzy c-means (FCM) clustering were also used to develop models for predicting E. coli. The performances of the predictive models were evaluated and compared using root mean squared log error (RMSLE). Cross-validation and model performance results indicated that although the majority of models predicted E. coli accurately, ANFIS models resulted in fewer errors compared to the other models. The ANFIS models have the potential to be used to predict E. coli concentration for intervention plans and monitoring programs for cascading dams, and to implement effective best management practices for grazing and irrigation during the growing season.

Fate and transport of antibiotics and antibiotic resistance genes in runoff and soil as affected by the timing of swine manure slurry application.

Land application of swine manure slurry is a common practice to supplement nutrients to soil for crop production. This practice can introduce antibiotic residues and antibiotic resistance genes (ARGs) into the environment. Field testing is critical in identifying manure management practices effective in minimizing the environmental impacts of manure-borne antibiotic and ARGs. The objective of this study was to determine how the timing of swine manure application relative to rainfall events impacts the fate and transport of antibiotics and ARGs in surface runoff and manure-amended soil. Swine manure slurry was either broadcast or injected on test plots in the field. A set of three 30-min simulated rainfall events, 24 h apart, were initiated on manured plots 1 day, 1 week, 2 weeks, or 3 weeks after the manure application. Results showed that an interval longer than 2 weeks between application and rainfall often significantly reduced the levels of antibiotics and ARGs tested in runoff with the exception of tet(X). For soil samples from broadcast plots, concentrations of two of the three antibiotics tested (lincomycin and tiamulin) decreased substantially in the first two weeks after manure application. In contrast, concentrations of most of the ARGs tested (tet(Q), tet(X), and erm(A)) in soil did not change significantly during the test period. Information obtained from the study can be beneficial in designing manure management practices and estimating the environmental loading of antibiotics and ARGs resulting from manure application.

Enhanced biodegradation of atrazine at high infiltration rates in agricultural soils.

The objective of this study was to assess the persistence and transport of atrazine at high infiltration rates expected from higher intensity precipitation associated with climate change scenarios in the midwestern U.S. The transport and transformation of atrazine was monitored in column experiments at high infiltration rates (64-119 mm d-1) associated with increased precipitation intensity. The optimum linear sorption and the lumped Monod biokinetic parameters were determined by inverting observed break-through curves (BTCs) using the advection-dispersion-sorption-degradation model. Batch microcosm studies were also conducted to examine the effect of moisture content (5%, 15% and 25%) on atrazine degradation and support the column results. BTCs from both soil types with continuous atrazine input showed a characteristic pattern of a pulse input i.e. lag phase prior to rapid atrazine degradation. The rate of atrazine leaching at higher infiltration rates was not fast enough to counteract the effect of enhanced degradation. Higher infiltration rates enriched the distribution of hydroxyatrazine in the soil profile for sandy loam, but their effect was minimal in loam soil. The pattern of degradation obtained in batch microcosms agreed with the column results. In both soils, mean half-life of atrazine was lower (4-8 days) at high soil moisture contents. Under future climate change scenarios, where more intense precipitation is likely to result in higher infiltration rates and increased soil moisture, the potential for groundwater pollution from atrazine may be reduced, especially in areas with a long history of atrazine application to soil.

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.

Microbial communities in the rhizosphere and the root of lettuce as affected by Salmonella-contaminated irrigation water.

Reclaimed wastewater is increasingly used as a source of irrigation water in croplands. The enteric pathogens in reclaimed wastewater may accumulate in soil and plants and cause food safety concerns. The objective of this study was to determine the effects of irrigation water containing Salmonella on the microbial communities in the rhizosphere and in the root of lettuce. The effects were also examined with three variables (soil texture, lettuce cultivar and harvest time) in a factorial design. Analyses of the 16S rRNA gene sequences show that the microbial communities in the root were significantly different from those in the rhizosphere, although ∼80% of the microbes in the root originated from the rhizosphere. Salmonella in irrigation water significantly altered the structure of the microbial community in the rhizosphere, but not in the root. Salmonella internalized in lettuce root was observed when contaminated water was used for irrigation. Compared to lettuce cultivar and harvest time, soil texture played a more significant role in shaping the bacterial communities in the rhizosphere and in the root. Results from this study could advance understanding about the long-term impact of reclaimed wastewater as a source of irrigation water on the microbiota associated with leafy green vegetables.

The Fate of Synthetic and Endogenous Hormones Used in the US Beef and Dairy Industries and the Potential for Human Exposure.

PURPOSE OF REVIEW: Growth-enhancing chemicals used by the beef and dairy industries may be bioavailable to humans via milk, meat, and other environmental matrices. This review evaluates the potential for environmental transport and bioavailability of the active chemical to humans. RECENT FINDINGS: Bovine somatostatin is detectable in milk; however, there is no evidence that the protein persists in the environment nor that it is active in humans. In contrast, steroids are transported through milk and meat to humans where they may exert biological activity. Furthermore, environmental matrices such as raw water and dust may also allow for the environmental transport and bioavailability of steroids to humans. Endogenous and exogenous steroids can be found in the meat, milk, and waste materials produced by cattle. While the concentrations may be low, exposure to these matrices, most notably dairy products made with whole milk, can be a source of exogenous steroids to humans.

Adsorption and decontamination of α-synuclein from medically and environmentally-relevant surfaces.

The assembly and accumulation of α-synuclein fibrils are implicated in the development of several neurodegenerative disorders including multiple system atrophy and Parkinson's disease. Pre-existing α-synuclein fibrils can recruit and convert soluble non-fibrillar α-synuclein to the fibrillar form similar to what is observed in prion diseases. This raises concerns regarding attachment of fibrillary α-synuclein to medical instruments and subsequent exposure of patients to α-synuclein similar to what has been observed in iatrogenic transmission of prions. Here, we evaluated adsorption and desorption of α-synuclein to two surfaces: stainless steel and a gold surface coated with a 11-Amino-1-undecanethiol hydrochloride self-assembled-monolayer (SAM) using in-situ combinatorial quartz crystal microbalance with dissipation and spectroscopic ellipsometry. α-Synuclein was found to attach to both surfaces, however, increased α-synuclein adsorption was observed onto the positively charged SAM surface compared to the stainless steel surface. Dynamic light scattering data showed that larger α-synuclein fibrils were preferentially attached to the stainless steel surface when compared with the distributions in the original α-synuclein solution and on the SAM surface. We determined that after attachment, introduction of a 1N NaOH solution could completely remove α-synuclein adsorbed on the stainless steel surface while α-synuclein was retained on the SAM surface. Our results indicate α-synuclein can bind to multiple surface types and that decontamination is surface-dependent.

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.

Response and recovery of fathead minnows (Pimephales promelas) following early life exposure to water and sediment found within agricultural runoff from the Elkhorn River, Nebraska, USA.

Agricultural runoff is a non-point source of chemical contaminants that are seasonally detected in surface water and sediments. Agrichemicals found within seasonal runoff can elicit endocrine disrupting effects in organisms as adults, juveniles and larvae. The objectives of this study were (1) to determine if exposure to water, sediment or the water-sediment combination collected from an agricultural runoff event was responsible for changes in endocrine-responsive gene expression and development in fathead minnow larvae, and (2) whether such early life exposure leads to adverse effects as adults. Larvae were exposed during the first month post-hatch to water and sediment collected from the Elkhorn River and then allowed to depurate in filtered water until reaching sexual maturity, exemplifying a best-case recovery scenario. Gas chromatography mass spectrometry (GC/MS) analysis of the water and sediment samples detected 12 pesticides including atrazine, acetochlor, metolachlor and dimethenamid. In minnow larvae, exposure to river water upregulated androgen receptor gene expression whereas exposure to the sediment downregulated estrogen receptor α expression. Adult males previously exposed to both water and sediment were feminized through the induction of an ovipositor structure whereas no impacts were observed in other reproductive or sex characteristic endpoints for either sex based on exposure history. Results from this study indicate that both water and sediments found in agricultural runoff elicit responses from minnow larvae, and larvae can recover following early life exposure under a best-case scenario.

Compensatory response of fathead minnow larvae following a pulsed in-situ exposure to a seasonal agricultural runoff event.

Agriculturally-dominated waterways such as those found throughout the Midwestern United States often experience seasonal pulses of agrichemical contaminants which pose a potential hazard to aquatic organisms at varying life stages. The objective of this study was to characterize the developmental plasticity of fathead minnow larvae in a natural environment subject to a seasonal episodic perturbation in the form of a complex mixture of agricultural stressors. Fathead minnow larvae were maintained at the Elkhorn River Research Station for a 28-d in situ exposure to an agrichemical pulse event. Minnow larvae were sampled after 14 and 28days to characterize developmental plasticity through growth measures and relative gene expression. Concentrations of agrichemical contaminants measured in water using polar organic chemical integrative samplers and composite sediment samples throughout the 28-d exposure were quantified using gas chromatography-mass spectrometry. Elevated concentrations of acetochlor, atrazine, and metolachlor were indicative of inputs from agricultural sources and were associated with reductions in body mass, condition factor, and androgenic gene expression in river exposed fathead minnow larvae. However, following a 14-d in situ depuration during the post-pulse period, river exposed larvae overcompensated in previously suppressed biological endpoints. These results indicate that fathead minnow larvae are capable of compensatory responses following episodic exposure to agrichemical stressors.