Infectivity of Wild-Bird Origin Influenza A Viruses in Minnesota Wetlands across Seasons.

The environmental tenacity of influenza A viruses (IAVs) in the environment likely plays a role in their transmission; IAVs are able to remain infectious in aquatic habitats and may have the capacity to seed outbreaks when susceptible wild bird hosts utilize these same environments months or even seasons later. Here, we aimed to assess the persistence of low-pathogenicity IAVs from naturally infected ducks in Northwestern Minnesota through a field experiment. Viral infectivity was measured using replicate samples maintained in distilled water in a laboratory setting as well as in filtered water from four natural water bodies maintained in steel perforated drums (hereafter, mesocosms) within the field from autumn 2020 to spring 2021. There was limited evidence for the extended persistence of IAVs held in mesocosms; from 65 initial IAV-positive samples, only six IAVs persisted to at least 202 days in the mesocosms compared to 17 viruses persisting at least this long when held under temperature-controlled laboratory settings in distilled water. When accounting for the initial titer of samples, viruses detected at a higher concentration at the initiation of the experiment persisted longer than those with a lower starting titer. A parallel experimental laboratory model was used to further explore the effects of water type on viral persistence, and the results supported the finding of reduced tenacity of IAVs held in mesocosms compared to distilled water. The results of this investigation provide evidence that many factors, including temperature and physicochemical properties, impact the duration of viral infectivity in natural settings, further extending our understanding of the potential and limitations of environmental-based methodologies to recover infectious IAVs.

Environmental Surveillance and Detection of Infectious Highly Pathogenic Avian Influenza Virus in Iowa Wetlands.

Avian influenza viruses (AIVs) infect both wild birds and domestic poultry, resulting in economically costly outbreaks that have the potential to impact public health. Currently, a knowledge gap exists regarding the detection of infectious AIVs in the aquatic environment. In response to the 2021-2022 Eurasian strain highly pathogenic avian influenza (HPAI) A/goose/Guangdong/1/1996 clade 2.3.4.4 lineage H5 outbreak, an AIV environmental outbreak response study was conducted using a One Health approach. An optimized method was used to temporally sample (April and May 2022) and analyze (culture and molecular methods) surface water from five water bodies (four wetlands and one lake used as a comparison location) in areas near confirmed HPAI detections in wild bird or poultry operations. Avian influenza viruses were isolated from water samples collected in April from all four wetlands (not from the comparison lake sample); HPAI H5N1 was isolated from one wetland. No virus was isolated from the May samples. Several factors, including increased water temperatures, precipitation, biotic and abiotic factors, and absence of AIV-contaminated fecal material due to fewer waterfowl present, may have contributed to the lack of virus isolation from May samples. Results demonstrate surface water as a plausible medium for transmission of AIVs, including the HPAI virus.

Simultaneous stream assessment of antibiotics, bacteria, antibiotic resistant bacteria, and antibiotic resistance genes in an agricultural region of the United States.

Antimicrobial resistance (AMR) is now recognized as a leading global threat to human health. Nevertheless, there currently is a limited understanding of the environment's role in the spread of AMR and antibiotic resistance genes (ARGs). In 2019, the U.S. Geological Survey conducted the first statewide assessment of antibiotic resistant bacteria (ARB) and ARGs in surface water and bed sediment collected from 34 stream locations across Iowa. Environmental samples were analyzed for a suite of 29 antibiotics and plated on selective media for 15 types of bacteria growth; DNA was extracted from culture growth and used in downstream polymerase chain reaction (PCR) assays for the detection of 24 ARGs. ARGs encoding resistance to antibiotics of clinical importance to human health and disease prevention were prioritized as their presence in stream systems has the potential for environmental significance. Total coliforms, Escherichia coli (E. coli), and staphylococci were nearly ubiquitous in both stream water and stream bed sediment samples, with enterococci present in 97 % of water samples, and Salmonella spp. growth present in 94 % and 67 % of water and bed sediment samples. Bacteria enumerations indicate that high bacteria loads are common in Iowa's streams, with 23 (68 %) streams exceeding state guidelines for primary contact for E. coli in recreational waters and 6 (18 %) streams exceeding the secondary contact advisory level. Although antibiotic-resistant E. coli growth was detected from 40 % of water samples, vancomycin-resistant enterococci (VRE) and penicillinase-resistant Staphylococcus aureus (MRSA) colony growth was detected from nearly all water samples. A total of 14 different ARGs were detected from viable bacteria cells from 30 Iowa streams (88 %, n = 34). Study results provide the first baseline understanding of the prevalence of ARB and ARGs throughout Iowa's waterways and health risk potential for humans, wildlife, and livestock using these waterways for drinking, irrigating, or recreating.

Contaminant Exposure and Transport from Three Potential Reuse Waters within a Single Watershed.

Global demand for safe and sustainable water supplies necessitates a better understanding of contaminant exposures in potential reuse waters. In this study, we compared exposures and load contributions to surface water from the discharge of three reuse waters (wastewater effluent, urban stormwater, and agricultural runoff). Results document substantial and varying organic-chemical contribution to surface water from effluent discharges (e.g., disinfection byproducts [DBP], prescription pharmaceuticals, industrial/household chemicals), urban stormwater (e.g., polycyclic aromatic hydrocarbons, pesticides, nonprescription pharmaceuticals), and agricultural runoff (e.g., pesticides). Excluding DBPs, episodic storm-event organic concentrations and loads from urban stormwater were comparable to and often exceeded those of daily wastewater-effluent discharges. We also assessed if wastewater-effluent irrigation to corn resulted in measurable effects on organic-chemical concentrations in rain-induced agricultural runoff and harvested feedstock. Overall, the target-organic load of 491 g from wastewater-effluent irrigation to the study corn field during the 2019 growing season did not produce substantial dissolved organic-contaminant contributions in subsequent rain-induced runoff events. Out of the 140 detected organics in source wastewater-effluent irrigation, only imidacloprid and estrone had concentrations that resulted in observable differences between rain-induced agricultural runoff from the effluent-irrigated and nonirrigated corn fields. Analyses of pharmaceuticals and per-/polyfluoroalkyl substances in at-harvest corn-plant samples detected two prescription antibiotics, norfloxacin and ciprofloxacin, at concentrations of 36 and 70 ng/g, respectively, in effluent-irrigated corn-plant samples; no contaminants were detected in noneffluent irrigated corn-plant samples.

Food, Beverage, and Feedstock Processing Facility Wastewater: a Unique and Underappreciated Source of Contaminants to U.S. Streams.

Process wastewaters from food, beverage, and feedstock facilities, although regulated, are an under-investigated environmental contaminant source. Food process wastewaters (FPWWs) from 23 facilities in 17 U.S. states were sampled and documented for a plethora of chemical and microbial contaminants. Of the 576 analyzed organics, 184 (32%) were detected at least once, with concentrations as large as 143 µg L-1 (6:2 fluorotelomer sulfonic acid), and as many as 47 were detected in a single FPWW sample. Cumulative per/polyfluoroalkyl substance concentrations up to 185 µg L-1 and large pesticide transformation product concentrations (e.g., methomyl oxime, 40 µg L-1; clothianidin TMG, 2.02 µg L-1) were observed. Despite 48% of FPWW undergoing disinfection treatment prior to discharge, bacteria resistant to third-generation antibiotics were found in each facility type, and multiple bacterial groups were detected in all samples, including total coliforms. The exposure-activity ratios and toxicity quotients exceeded 1.0 in 13 and 22% of samples, respectively, indicating potential biological effects and toxicity to vertebrates and invertebrates associated with the discharge of FPWW. Organic contaminant profiles of FPWW differed from previously reported contaminant profiles of municipal effluents and urban storm water, indicating that FPWW is another important source of chemical and microbial contaminant mixtures discharged into receiving surface waters.

Evidence for interannual persistence of infectious influenza A viruses in Alaska wetlands.

Influenza A viruses (IAVs) deposited by wild birds into the environment may lead to sporadic mortality events and economically costly outbreaks among domestic birds. There is a paucity of information, however, regarding the persistence of infectious IAVs within the environment following deposition. In this investigation, we assessed the persistence of 12 IAVs that were present in cloacal and/or oropharyngeal swabs of naturally infected ducks. Infectivity of these IAVs was monitored over approximately one year with each virus tested in five water types: (1) distilled water held in the lab at 4 °C and (2-5) filtered surface water from each of four Alaska sites and maintained in the field at ambient temperature. By evaluating infectivity of IAVs in ovo following sample retrieval at four successive time points, we observed declines in IAV infectivity through time. Many viruses persisted for extended periods, as evidenced by ≥25% of IAVs remaining infectious in replicate samples for each treatment type through three sampling time points (144-155 days post-sample collection) and two viruses remaining viable in a single replicate sample each when tested upon collection at a fourth time point (361-377 days post-sample collection). The estimated probability of persistence of infectious IAVs in all five water types was estimated to be between 0.25 and 0.75 during days 50-200 post-sample collection as inferred through Kaplan-Meier survival analysis. Our results provide evidence that IAVs may remain infectious for extended periods, up to or even exceeding one year, when maintained in surface waters under ambient temperatures. Therefore, wetlands may represent an important medium in which infectious IAVs may reside outside of a biotic reservoir.

Exposure and Transport of Alkaloids and Phytoestrogens from Soybeans to Agricultural Soils and Streams in the Midwestern United States.

Phytotoxins are naturally produced toxins with potencies similar/higher than many anthropogenic micropollutants. Nevertheless, little is known regarding their environmental fate and off-field transport to streams. To fill this research gap, a network of six basins in the Midwestern United States with substantial soybean production was selected for the study. Stream water (n = 110), soybean plant tissues (n = 8), and soil samples (n = 16) were analyzed for 12 phytotoxins (5 alkaloids and 7 phytoestrogens) and 2 widely used herbicides (atrazine and metolachlor). Overall, at least 1 phytotoxin was detected in 82% of the samples, with as many as 11 phytotoxins detected in a single sample (median = 5), with a concentration range from below detection to 37 and 68 ng/L for alkaloids and phytoestrogens, respectively. In contrast, the herbicides were ubiquitously detected at substantially higher concentrations (atrazine: 99% and metolachlor: 83%; the concentrations range from below detection to 150 and 410 ng/L, respectively). There was an apparent seasonal pattern for phytotoxins, where occurrence prior to and during harvest season (September to November) and during the snow melt season (March) was higher than that in December-January. Runoff events increased phytotoxin and herbicide concentrations compared to those in base-flow conditions. Phytotoxin plant concentrations were orders of magnitude higher compared to those measured in soil and streams. These results demonstrate the potential exposure of aquatic and terrestrial organisms to soybean-derived phytotoxins.

Highlighting the complexities of a groundwater pilot study during an avian influenza outbreak: Methods, lessons learned, and select contaminant results.

The highly pathogenic avian influenza (H5N2) outbreak in the Midwestern United States (US) in 2015 was historic due to the number of birds and poultry operations impacted and the corresponding economic loss to the poultry industry and was the largest animal health emergency in US history. The U.S. Geological Survey (USGS), with the assistance of several state and federal agencies, aided the response to the outbreak by developing a study to determine the extent of virus transport in the environment. The study goals were to: develop the appropriate sampling methods and protocols for measuring avian influenza virus (AIV) in groundwater, provide the first baseline data on AIV and outbreak- and poultry-related contaminant occurrence and movement into groundwater, and document climatological factors that may have affected both survival and transport of AIV to groundwater during the months of the 2015 outbreak. While site selection was expedient, there were often delays in sample response times due to both relationship building between agencies, groups, and producers and logistical time constraints. This study's design and sampling process highlights the unpredictable nature of disease outbreaks and the corresponding difficulty in environmental sampling of such events. The lessons learned, including field protocols and approaches, can be used to improve future research on AIV in the environment.

Pre/post-closure assessment of groundwater pharmaceutical fate in a wastewater-facility-impacted stream reach.

Pharmaceutical contamination of contiguous groundwater is a substantial concern in wastewater-impacted streams, due to ubiquity in effluent, high aqueous mobility, designed bioactivity, and to effluent-driven hydraulic gradients. Wastewater treatment facility (WWTF) closures are rare environmental remediation events; offering unique insights into contaminant persistence, long-term wastewater impacts, and ecosystem recovery processes. The USGS conducted a combined pre/post-closure groundwater assessment adjacent to an effluent-impacted reach of Fourmile Creek, Ankeny, Iowa, USA. Higher surface-water concentrations, consistent surface-water to groundwater concentration gradients, and sustained groundwater detections tens of meters from the stream bank demonstrated the importance of WWTF effluent as the source of groundwater pharmaceuticals as well as the persistence of these contaminants under effluent-driven, pre-closure conditions. The number of analytes (110 total) detected in surface water decreased from 69 prior to closure down to 8 in the first post-closure sampling event approximately 30 d later, with a corresponding 2 order of magnitude decrease in the cumulative concentration of detected analytes. Post-closure cumulative concentrations of detected analytes were approximately 5 times higher in proximal groundwater than in surface water. About 40% of the 21 contaminants detected in a downstream groundwater transect immediately before WWTF closure exhibited rapid attenuation with estimated half-lives on the order of a few days; however, a comparable number exhibited no consistent attenuation during the year-long post-closure assessment. The results demonstrate the potential for effluent-impacted shallow groundwater systems to accumulate pharmaceutical contaminants and serve as long-term residual sources, further increasing the risk of adverse ecological effects in groundwater and the near-stream ecosystem.

Genes Indicative of Zoonotic and Swine Pathogens Are Persistent in Stream Water and Sediment following a Swine Manure Spill.

Manure spills into streams are relatively frequent, but no studies have characterized stream contamination with zoonotic and veterinary pathogens, or fecal chemicals, following a spill. We tested stream water and sediment over 25 days and downstream for 7.6 km for the following: fecal indicator bacteria (FIB), the fecal indicator chemicals cholesterol and coprostanol, 20 genes for zoonotic and swine-specific bacterial pathogens by presence/absence PCR for viable cells, one swine-specific Escherichia coli toxin gene (STII gene) by quantitative PCR (qPCR), and nine human and animal viruses by qPCR or reverse transcription-qPCR. Twelve days postspill, and 4.2 km downstream, water concentrations of FIB, cholesterol, and coprostanol were 1 to 2 orders of magnitude greater than those detected before, or above, the spill, and genes indicating viable zoonotic or swine-infectious Escherichia coli were detected in water or sediment. STII gene levels increased from undetectable before or above the spill to 10(5) copies/100 ml of water 12 days postspill. Thirteen of 14 water (8/9 sediment) samples had viable STII-carrying cells postspill. Eighteen days postspill, porcine adenovirus and teschovirus were detected 5.6 km downstream. FIB concentrations (per gram [wet weight]) in sediment were greater than in water, and sediment was a continuous reservoir of genes and chemicals postspill. Constituent concentrations were much lower, and detections less frequent, in a runoff event (200 days postspill) following manure application, although the swine-associated STII and stx2e genes were detected. Manure spills are an underappreciated pathway for livestock-derived contaminants to enter streams, with persistent environmental outcomes and the potential for human and veterinary health consequences.

Riverbank filtration potential of pharmaceuticals in a wastewater-impacted stream.

Pharmaceutical contamination of shallow groundwater is a substantial concern in effluent-dominated streams, due to high aqueous mobility, designed bioactivity, and effluent-driven hydraulic gradients. In October and December 2012, effluent contributed approximately 99% and 71%, respectively, to downstream flow in Fourmile Creek, Iowa, USA. Strong hydrologic connectivity was observed between surface-water and shallow-groundwater. Carbamazepine, sulfamethoxazole, and immunologically-related compounds were detected in groundwater at greater than 0.02 µg L(-1) at distances up to 6 m from the stream bank. Direct aqueous-injection HPLC-MS/MS revealed 43% and 55% of 110 total pharmaceutical analytes in surface-water samples in October and December, respectively, with 16% and 6%, respectively, detected in groundwater approximately 20 m from the stream bank. The results demonstrate the importance of effluent discharge as a driver of local hydrologic conditions in an effluent-impacted stream and thus as a fundamental control on surface-water to groundwater transport of effluent-derived pharmaceutical contaminants.

Mycotoxins: diffuse and point source contributions of natural contaminants of emerging concern to streams.

To determine the prevalence of mycotoxins in streams, 116 water samples from 32 streams and three wastewater treatment plant effluents were collected in 2010 providing the broadest investigation on the spatial and temporal occurrence of mycotoxins in streams conducted in the United States to date. Out of the 33 target mycotoxins measured, nine were detected at least once during this study. The detections of mycotoxins were nearly ubiquitous during this study even though the basin size spanned four orders of magnitude. At least one mycotoxin was detected in 94% of the 116 samples collected. Deoxynivalenol was the most frequently detected mycotoxin (77%), followed by nivalenol (59%), beauvericin (43%), zearalenone (26%), ß-zearalenol (20%), 3-acetyl-deoxynivalenol (16%), α-zearalenol (10%), diacetoxyscirpenol (5%), and verrucarin A (1%). In addition, one or more of the three known estrogenic compounds (i.e. zearalenone, α-zearalenol, and ß-zearalenol) were detected in 43% of the samples, with maximum concentrations substantially higher than observed in previous research. While concentrations were generally low (i.e. < 50 ng/L) during this study, concentrations exceeding 1,000 ng/L were measured during spring snowmelt conditions in agricultural settings and in wastewater treatment plant effluent. Results of this study suggest that both diffuse (e.g. release from infected plants and manure applications from exposed livestock) and point (e.g. wastewater treatment plants and food processing plants) sources are important environmental pathways for mycotoxin transport to streams. The ecotoxicological impacts from the long-term, low-level exposures to mycotoxins alone or in combination with complex chemical mixtures are unknown.

Chemical contaminants in water and sediment near fish nesting sites in the Potomac River basin: determining potential exposures to smallmouth bass (Micropterus dolomieu).

The Potomac River basin is an area where a high prevalence of abnormalities such as testicular oocytes (TO), skin lesions, and mortality has been observed in smallmouth bass (SMB, Micropterus dolomieu). Previous research documented a variety of chemicals in regional streams, implicating chemical exposure as one plausible explanation for these biological effects. Six stream sites in the Potomac basin (and one out-of-basin reference site) were sampled to provide an assessment of chemicals in these streams. Potential early life-stage exposure to chemicals detected was assessed by collecting samples in and around SMB nesting areas. Target chemicals included those known to be associated with important agricultural and municipal wastewater sources in the Potomac basin. The prevalence and severity of TO in SMB were also measured to determine potential relations between chemistry and biological effects. A total of 39 chemicals were detected at least once in the discrete-water samples, with atrazine, caffeine, deethylatrazine, simazine, and iso-chlorotetracycline being most frequently detected. Of the most frequently detected chemicals, only caffeine was detected in water from the reference site. No biogenic hormones/sterols were detected in the discrete-water samples. In contrast, 100 chemicals (including six biogenic hormones/sterols) were found in a least one passive-water sample, with 25 being detected at all such samples. In addition, 46 chemicals (including seven biogenic hormones/sterols) were found in the bed-sediment samples, with caffeine, cholesterol, indole, para-cresol, and sitosterol detected in all such samples. The number of herbicides detected in discrete-water samples per site had a significant positive relation to TO(rank) (a nonparametric indicator of TO), with significant positive relations between TO(rank) and atrazine concentrations in discrete-water samples and to total hormone/sterol concentration in bed-sediment samples. Such significant correlations do not necessarily imply causation, as these chemical compositions and concentrations likely do not adequately reflect total SMB exposure history, particularly during critical life stages.

Phytoestrogens and mycotoxins in Iowa streams: an examination of underinvestigated compounds in agricultural basins.

This study provides the first broad-scale investigation on the spatial and temporal occurrence of phytoestrogens and mycotoxins in streams in the United States. Fifteen stream sites across Iowa were sampled five times throughout the 2008 growing season to capture a range of climatic and crop-growth conditions. Basin size upstream from sampling sites ranged from 7 km2 to > 836,000 km2. Atrazine (herbicide) also was measured in all samples as a frame-of-reference agriculturally derived contaminant. Target compounds were frequently detected in stream samples: atrazine (100%), formononetin (80%), equol (45%), deoxynivalenol (43%), daidzein (32%), biochanin A (23%), zearalenone (13%), and genistein (11%). The nearly ubiquitous detection of formononetin (isoflavone) suggests a widespread agricultural source, as one would expect with the intense row crop and livestock production present across Iowa. Conversely, the less spatially widespread detections of deoxynivalenol (mycotoxin) suggest a more variable source due to the required combination of proper host and proper temperature and moisture conditions necessary to promote Fusarium spp. infections. Although atrazine concentrations commonly exceeded 100 ng L(-1) (42/75 measurements), only deoxynivalenol (6/56 measurements) had concentrations that occasionally exceeded this level. Temporal patterns in concentrations varied substantially between atrazine, formononetin, and deoxynivalenol, as one would expect for contaminants with different source inputs and processes of formation and degradation. The greatest phytoestrogen and mycotoxin concentrations were observed during spring snowmelt conditions. Phytoestrogens and mycotoxins were detected at all sampling sites regardless of basin size. The ecotoxicological effects from long-term, low-level exposures to phytoestrogens and mycotoxins or complex chemicals mixtures including these compounds that commonly rake place in surface water are poorly understood and have yet to be systematically investigated in environmental studies.

Impact of sampling strategy on stream load estimates in till landscape of the Midwest.

Accurately estimating various solute loads in streams during storms is critical to accurately determine maximum daily loads for regulatory purposes. This study investigates the impact of sampling strategy on solute load estimates in streams in the US Midwest. Three different solute types (nitrate, magnesium, and dissolved organic carbon (DOC)) and three sampling strategies are assessed. Regardless of the method, the average error on nitrate loads is higher than for magnesium or DOC loads, and all three methods generally underestimate DOC loads and overestimate magnesium loads. Increasing sampling frequency only slightly improves the accuracy of solute load estimates but generally improves the precision of load calculations. This type of investigation is critical for water management and environmental assessment so error on solute load calculations can be taken into account by landscape managers, and sampling strategies optimized as a function of monitoring objectives.