Investigating the partitioning behavior of per- and polyfluoroalkyl substances (PFAS) during thermal landfill leachate evaporation.

Thermal landfill leachate evaporator systems can reduce the volume of leachate by up to 97%, while releasing water vapor and producing residuals (volume-reduced leachate and sludge) that are managed on-site. On-site thermal evaporators offer landfill operators leachate management autonomy without being subject to increasingly stringent wastewater treatment plant requirements. However, little is known about the partitioning of PFAS within these systems, nor the extent to which PFAS may be emitted into the environment via vapor. In this study, feed leachate, residual evaporated leachate, sludge, and condensed vapor were sampled at two active full-scale thermal landfill leachate evaporators and from a laboratory-scale leachate evaporation experiment. Samples were analyzed for 91 PFAS via ultra-high pressure liquid chromatography - tandem mass spectrometry (UHPLC-MS/MS). Similar trends were observed from Evaporator 1, Evaporator 2, and the laboratory-scale evaporator; ∑PFAS were concentrated in the residual evaporated leachate during evaporation by a factor of 5.3 to 20. All condensed vapors sampled (n = 5) contained PFAS, predominantly 5:3 fluorotelomer carboxylic acid (5:3FTCA), (full-scale vapors 729 - 4087 ng/L PFAS; lab-scale vapor 61.0 ng/L PFAS). For Evaporators 1 and 2, an estimated 9 - 24% and 10%, respectively, of the PFAS mass entering the evaporators in leachate was released with vapor during the days of sample collection. '.

Characterization of per- and polyfluoroalkyl substances (PFAS) and other constituents in MSW landfill leachate from Puerto Rico.

Elevated per- and polyfluoroalkyl substance (PFAS) concentrations have been reported in municipal solid waste (MSW) landfill leachate with higher levels in wet and warmer subtropical climates. Information about landfill leachate characteristics is much more limited in tropical climates. In this study, 20 landfill leachate samples were collected from three MSW landfills on the tropical island of Puerto Rico and results were compared against landfills nationally and within Florida, USA. The samples collected in Puerto Rico underwent physical-chemical analysis, as well as a quantitative analysis of 92 PFAS. Samples described in this study include discrete leachate types, such as leachate, gas condensate, and leachate which has undergone on-site treatment (e.g., RO treatment, phytoremediation, lagoons). A total of 51 PFAS were detected above quantitation limits, including perfluorohexylphosphonic acid, a perfluoroalkyl acid (PFAA) which has not been reported previously in landfill leachate. ∑PFAS concentrations in this study (mean: 38,000 ng L-1), as well as concentrations of individual PFAS, are significantly higher than other reported MSW landfill leachate concentrations. The profiles of leachates collected from on-site treatment systems indicate possible transformation of precursor PFAS as a result of treatment processes - oxidizing conditions, for example, may facilitate aerobic transformation, increase the concentrations of PFAAs, and possibly increase the apparent ∑PFAS concentration. Extreme climate events, including rising temperatures and more frequent hurricanes, have placed additional strain on the solid waste management infrastructure on the island - adding complexity to an already challenging PFAS management issue. As concern grows over PFAS contamination in drinking water, these findings should inform solid waste and leachate management decisions in order to minimize PFAS emissions in island environments.

Evaluation of per- and polyfluoroalkyl substances (PFAS) in landfill liquids from Pennsylvania, Colorado, and Wisconsin.

PER: and polyfluoroalkyl substances (PFAS) have been measured in aqueous components within landfills. To date, the majority of these studies have been conducted in Florida. This current study aimed to evaluate PFAS concentrations in aqueous components (leachate, gas condensate, stormwater, and groundwater) from four landfills located outside of Florida, in Pennsylvania, Colorado, and Wisconsin (2 landfills). The Pennsylvania landfill also provided the opportunity to assess a leachate treatment system. Sample analyses were consistent across studies including the measurements of 26 PFAS and physical-chemical parameters. For the four target landfills, average PFAS concentrations were 6,900, 22,000, 280, and 260 ng L-1 in the leachate, gas condensate, stormwater, and groundwater, respectively. These results were not significantly different than those observed for landfills in Florida except for the significantly higher PFAS concentrations in gas condensate compared to leachate. For on-site treatment at the Pennsylvania landfill, results suggest that the membrane biological bioreactor (MBBR) system performed similarly as aeration-based leachate treatment systems at Florida landfills resulting in no significant decreases in ∑26PFAS. Overall, results suggest a general consistency across US regions in PFAS concentrations within different landfill liquid types, with the few differences observed likely influenced by landfill design and local climate. Results confirm that leachate exposed to open air (e.g., in trenches or in treatment systems) have lower proportions of perfluoroalkyl acid precursors relative to leachate collected in enclosed pipe systems. Results also confirm that landfills without bottom liner systems may have relatively higher PFAS levels in adjacent groundwater and that landfills in wetter climates tend to have higher PFAS concentrations in leachate.

Non-traditional abiotic drivers explain variability of chlorophyll-a in a shallow estuarine embayment.

Understanding the factors influencing eutrophication, as represented by concentrations of chlorophyll-a (Chl-a), is needed to inform effective management and conservation strategies promoting ecological resilience. The objective of this study was to evaluate a unique combination of abiotic explanatory factors to describe Chl-a concentrations within the study estuary (North Biscayne Bay, Florida, USA). Multiple linear regression determined the strength and direction of influence of factors using data from 10 water quality monitoring stations. The analysis also considered time scales for evaluating cumulative effects of freshwater inflow and wind. Results show that dominant drivers of Chl-a were temperature, freshwater volume (whose cumulative effects were evaluated up to a 60-day time scale), and turbidity, which were statistically significant at 60, 60, and 70 % of the investigated stations, respectively. All drivers collectively accounted for 22 to 63 % of the variability of Chl-a measurements. Of the nine variables evaluated, nutrient concentrations (orthophosphate and ammonia) were not among the top three overall drivers. Despite nutrients historically being cited in the literature as the most significant factor, this study asserts that non-nutrient factors often govern Chl-a levels, necessitating a paradigm shift in management strategies to bolster estuarine resilience against climate change.

Wastewater based surveillance can be used to reduce clinical testing intensity on a university campus.

Clinical testing has been a vital part of the response to and suppression of the COVID-19 pandemic; however, testing imposes significant burdens on a population. College students had to contend with clinical testing while simultaneously dealing with health risks and the academic pressures brought on by quarantines, changes to virtual platforms, and other disruptions to daily life. The objective of this study was to analyze whether wastewater surveillance can be used to decrease the intensity of clinical testing while maintaining reliable measurements of diseases incidence on campus. Twelve months of human health and wastewater surveillance data for eight residential buildings on a university campus were analyzed to establish how SARS-CoV-2 levels in the wastewater can be used to minimize clinical testing burden on students. Wastewater SARS-CoV-2 levels were used to create multiple scenarios, each with differing levels of testing intensity, which were compared to the actual testing volumes implemented by the university. We found that scenarios in which testing intensity fluctuations matched rise and falls in SARS-CoV-2 wastewater levels had stronger correlations between SARS-CoV-2 levels and recorded clinical positives. In addition to stronger correlations, most scenarios resulted in overall fewer weekly clinical tests performed. We suggest the use of wastewater surveillance to guide COVID-19 testing as it can significantly increase the efficacy of COVID-19 surveillance while reducing the burden placed on college students during a pandemic. Future efforts should be made to integrate wastewater surveillance into clinical testing strategies implemented on college campuses.

Evaluation of per- and polyfluoroalkyl substances (PFAS) released from two Florida landfills based on mass balance analyses.

Per- and polyfluoroalkyl substances (PFAS) have been found at high levels within landfill environments. To assess PFAS distributions, this study aimed to evaluate PFAS mass flux leached from disposed solid waste and within landfill reservoirs by mass balance analyses for two full-scale operational Florida landfills. PFAS mass flux in different aqueous components within landfills were estimated based on PFAS concentrations and water flow rates. For PFAS concentration, 26 PFAS, including 18 perfluoroalkyl acids (PFAAs) and 8 PFAA-precursors, were measured in samples collected from the landfills or estimated based on previous studies. Flow rates of aqueous components (rainfall, evapotranspiration, runoff, stormwater, groundwater, leakage, gas condensate, and leachate) were evaluated through the Hydrologic Evaluation of Landfill Performance model, water balance, and Darcy's Law. Results showed that the average PFAS mass flux leached from the solid waste standardized by area was estimated as 36.8 g/ha-yr, which was approximately 1 % to 3 % of the total amount of PFAS within the solid waste. The majority of PFAS leached from the solid waste (95 % to 97 %) is captured by the leachate collection system, with other aqueous components representing much smaller fractions (stormwater system at 3 % to 5 %, and gas condensate and groundwater at < 1 %). Also, based on the results, we estimate that PFAS releases will likely occur at least over 40 years. Overall, these results can help prioritize components for waste management and PFAS treatment during the anticipated landfill release periods.

Comparison of the PFAS and physical-chemical parameter fluctuations between an ash landfill and a MSW landfill.

Studies of per- and polyfluoroalkyl substances (PFAS) fluctuations at landfills have focused on municipal solid waste (MSW) leachate. Few studies exist that evaluate fluctuations (defined by the coefficient of variation, CV) in MSW incinerator ash (MSWA) landfill leachate and that evaluate PFAS fluctuations in stormwater, groundwater, and treated liquids on-site. In this study, aqueous landfill samples (leachate, treated leachate, stormwater, gas condensate, ambient groundwater, and effluent from a groundwater remediation system) were collected from a MSW and an MSWA landfill geographically located within close proximity (less than 40 km). The objective of this study was to compare the leachate compositions between these two landfill types and to evaluate temporal variations. Results indicated that the CV of total detected PFAS concentrations in leachate was higher for the MSW landfill (CV = 43 %) compared to the MSWA landfill (CV = 16 %). The total detected PFAS concentration in MSW leachate samples (mean: 9641 ng/L) was higher than in MSWA leachate samples (mean: 2621 ng/L) (p < 0.05). Within a landfill, PFAS concentrations were correlated (rs > 0.6, p < 0.05) with alkalinity, total organic carbon (TOC), and ammonia. Results from the on-site leachate treatment system at the MSW landfill indicated reductions in COD, TOC, and ammonia; however, the ∑26PFAS concentration increased 3 % after the treatment. Overall, results demonstrated that differences between landfill types and fluctuations in PFAS within landfills should be considered when designing landfill leachate collection and treatment systems to remove PFAS. The comparative analysis in this study can provide insights into optimizing leachate management for MSW and MSWA landfills.

Assessing quality and beneficial uses of Sargassum compost.

Sargassum spp. (specifically Sargassum fluitans and S. natans), one of the dominant forms of marine macroalgae (seaweed) found on the beaches of Florida, is washing up on the shores throughout the Caribbean in record quantities. Currently, a common management option is to haul and dispose of beached Sargassum in local landfills, potentially wasting a valuable renewable resource. The objective of this study was to determine whether composting represents a feasible alternative to managing Sargassum inundations through measurements and comparisons to eleven guidelines. Specifically, we assessed the characteristics of the compost [physical-chemical parameters (temperature, moisture content, pH, and conductivity), nutrient ratios (C:N), elemental composition, bacteria levels, and ability to sustain plant growth] in both small- and large scale experiments. Results show that although nutrient concentration ratios were not within the standards outlined by the U.S. Composting Council (USCC), the Sargassum compost was able to sustain the growth of radishes (Raphanus sativus L., var. Champion). Trace metal concentrations in the compost product were within five regulatory guidelines evaluated, except for arsenic (As) (6.64-26.5 mg/kg), which exceeded one of the five (the Florida Soil Cleanup Target Level for residential use). Bacteria levels were consistent with regulatory guidelines for compost produced in large-scale outdoor experiments but not for the small-scale set conducted in enclosed tumblers. Overall results support that Sargassum compost can be beneficially used for fill and some farming applications.

Geospatially-resolved public-health surveillance via wastewater sequencing.

Wastewater, which contains everything from pathogens to pollutants, is a geospatially-and temporally-linked microbial fingerprint of a given population. As a result, it can be leveraged for monitoring multiple dimensions of public health across locales and time. Here, we integrate targeted and bulk RNA sequencing (n=1,419 samples) to track the viral, bacterial, and functional content over geospatially distinct areas within Miami Dade County from 2020-2022. First, we used targeted amplicon sequencing (n=966) to track diverse SARS-CoV-2 variants across space and time, and we found a tight correspondence with clinical caseloads from University students (N = 1,503) and Miami-Dade County hospital patients (N = 3,939 patients), as well as an 8-day earlier detection of the Delta variant in wastewater vs. in patients. Additionally, in 453 metatranscriptomic samples, we demonstrate that different wastewater sampling locations have clinically and public-health-relevant microbiota that vary as a function of the size of the human population they represent. Through assembly, alignment-based, and phylogenetic approaches, we also detect multiple clinically important viruses (e.g., norovirus ) and describe geospatial and temporal variation in microbial functional genes that indicate the presence of pollutants. Moreover, we found distinct profiles of antimicrobial resistance (AMR) genes and virulence factors across campus buildings, dorms, and hospitals, with hospital wastewater containing a significant increase in AMR abundance. Overall, this effort lays the groundwork for systematic characterization of wastewater to improve public health decision making and a broad platform to detect emerging pathogens.

Strategies for Monitoring Microbial Life in Beach Sand for Protection of Public Health.

The 2021 revised guidelines of the World Health Organization recommend monitoring the quality of sand in addition to water at recreational beaches. This review provides background information about the types of beaches, the characteristics of sand, and the microbiological parameters that should be measured. Analytical approaches are described for quantifying fungi and fecal indicator bacteria from beach sand. The review addresses strategies to assess beach sand quality, monitoring approaches, sand remediation, and the proposed way forward for beach sand monitoring programs. In the proposed way forward, recommendations are provided for acceptable levels of fungi given their distribution in the environment. Additional recommendations include evaluating FIB distributions at beaches globally to assess acceptable ranges of FIB levels, similar to those proposed for fungi.

Monkeypox viral nucleic acids detected using both DNA and RNA extraction workflows.

Molecular methods have been used to detect human pathogens in wastewater with sampling typically performed at wastewater treatment plants (WWTP) and upstream locations within the sewer system. A wastewater-based surveillance (WBS) program was established at the University of Miami (UM) in 2020, which included measurements of SARS-CoV-2 levels in wastewater from its hospital and within the regional WWTP. In addition to the development of a SARS-CoV-2 quantitative PCR (qPCR) assay, qPCR assays to detect other human pathogens of interest were also developed at UM. Here we report on the use of a modified set of reagents published by the CDC to detect nucleic acids of Monkeypox virus (MPXV) which emerged during May of 2022 to become a concern worldwide. Samples collected from the University hospital and from the regional WWTP were processed through DNA and RNA workflows and analyzed by qPCR to detect a segment of the MPXV CrmB gene. Results show positive detections of MPXV nucleic acids in the hospital and wastewater treatment plant wastewater which coincided with clinical cases in the community and mirrored the overall trend of nationwide MPXV cases reported to the CDC. We recommend the expansion of current WBS programs' methods to detect a broader range of pathogens of concern in wastewater and present evidence that viral RNA in human cells infected by a DNA virus can be detected in wastewater.

Microbial communities in the water surface microlayer and associations with microbes in aerosols, beach sand, and bulk water.

The water surface microlayer (SML) serves as a boundary through which microbes can be exchanged. To evaluate exchanges of microbes, this study compared microbial communities within different reservoirs, with an emphasis on the water SML and aerosols. Additionally, the microbial communities during a sewage spill and perigean tides were evaluated and the results were compared to times without these events. Results show that during perigean tides and during the sewage spill, levels of culturable bacteria were highest and showed an increase via sequencing in potential pathogenic bacteria (Corynebacterium and Vibrio, which increased from 3.5%-1800% depending on sample type). In the aerosol samples, Corynebacterium (average of 2.0%), Vibrio (1.6%), and Staphylococcus (10%), were the most abundant genera. Aerosolization factors, which were used to examine the transfer of the microbes, were high for these three genera. Measurements of general marine bacteria (GMB) by culture showed a weak but significant correlation between culturable GMB in aerosol samples versus in water and in the SML. More research is needed to evaluate the exchange of pathogens between the SML and air, given the increase in potentially pathogenic microbes within the SML during rare events and evidence that suggests that microbes maintain viability during transfers across reservoirs.

Relationships between per- and polyfluoroalkyl substances (PFAS) and physical-chemical parameters in aqueous landfill samples.

Variable chemistries of liquids from landfills can potentially impact levels of per- and polyfluoroalkyl substances (PFAS). The objective of the current study was to evaluate relationships between physical-chemical properties (bulk measurements, oxygen demand components, and metals) and PFAS concentrations in different types of aqueous landfill samples. Aqueous landfill samples were collected from 39 landfill facilities in Florida, United States. These samples included leachates from landfills that receive different waste types, such as municipal solid waste incineration ash (MSWA), construction and demolition debris (C&D), and municipal solid waste (MSW). Additional aqueous landfill samples were sourced from treated landfill leachate, gas condensate, stormwater, and groundwater from within and near the landfill boundaries. Results showed significant correlations (p < 0.05) between ∑26PFAS and alkalinity (rs = 0.83), total organic carbon (TOC) (rs = 0.84), and ammonia (rs = 0.79) for all leachate types. Other physical-chemical parameters that were significantly correlated (rs > 0.60, p < 0.05) with PFAS included specific conductivity, chemical oxygen demand (COD), and to a lesser extent, total dissolved solids (TDS) and total solids (TS). For gas condensates, PFAS was significantly correlated with TOC. Stormwater and groundwater, within and near the landfill boundaries, had considerably lower levels of PFAS and had a minimal correlation between PFAS and physical-chemical parameters. Although PFAS concentrations and physical-chemical parameters and their correlations varied between different types of aqueous landfill samples, results suggest that physical-chemical properties can be useful indicators of relative PFAS concentrations within a leachate type. More research is needed to validate the mechanisms that relate physical-chemical parameters to PFAS concentrations in landfill leachates.

Underestimation of Per- and Polyfluoroalkyl Substances in Biosolids: Precursor Transformation During Conventional Treatment.

Wastewater treatment plants generate a solid waste known as biosolids. The most common management option for biosolids is to beneficially reuse them as an agricultural amendment, but because of the risk of pathogen exposure, many regulatory bodies require pathogen reduction before biosolids reuse. Per- and polyfluoroalkyl substances (PFAS) are well documented in biosolids, but limited information is available on how biosolids treatment processes impact PFAS. Furthermore, quantification of PFAS has focused on perfluoroalkyl acids (PFAAs) which are a small fraction of thousands of PFAS known to exist. The objective of this study was to quantify 92 PFAS in biosolids collected from eight biosolids treatment facilities before and after four pathogen treatment applications: composting, heat treatment, lime treatment, and anaerobic digestion. Overall, total PFAS concentrations before and after treatment were dominated by PFAA precursor species, in particular, diPAPs which accounted for a majority of the mass of the Σ92PFAS. This differs from historic data that found PFAAs, primarily PFOS, to dominate total PFAS concentrations. Treatment options such as heat treatment and composting changed the ratio of PFAA precursors to PFAAs indicating a transformation of PFAS during treatment. This study finds that PFAA precursors are likely underrepresented by other studies and make up a larger percentage of the total PFAS concentration in biosolids than previously estimated.

Per- and polyfluoroalkyl substances (PFAS) distribution in landfill gas collection systems: leachate and gas condensate partitioning.

While per- and polyfluoroalkyl substances (PFAS) have been reported extensively in municipal solid waste (MSW) landfill leachate,they have rarely been quantified in landfill gas or in discrete landfill liquids such as landfill gas condensate (LGC), and the potential for PFAS to partition to the condensate has not been reported. LGC and leachate collected from within gas wells known as gas well pump-out (GWP) from three MSW landfills underwent physical-chemical characterization and PFAS analysis to improve understanding of the conditions under which these liquids form and to illuminate PFAS behavior within landfills. LGC was observed to be clear liquid containing ammonia and alkalinity while GWP strongly resembled leachate - dark in color, high in chloride and ammonia. Notably, arsenic and antimony were found in concentrations exceeding regulatory thresholds by over two orders of magnitude in many LGC samples. LGC contained a lower average concentration of ΣPFAS (19,000 ng L) compared to GWP (56,000 ng L); however, LGC contained more diversity of PFAS, with 53 quantified compared to 44 in GWP. LGC contained proportionally more precursor PFAS than GWP, including more semi-volatile PFAS which are rarely measured in water matrices, such as fluorotelomer alcohols and perfluoroalkane sulfonamido ethanols. This study provides the first detailed comparison of these matrices to inform timely leachate management decisions.

Degradation rates influence the ability of composite samples to represent 24-hourly means of SARS-CoV-2 and other microbiological target measures in wastewater.

The utility of using severe-acute respiratory syndrome coronavirus-2 (SARS-CoV-2) RNA for assessing the prevalence of COVID-19 within communities begins with the design of the sample collection program. The objective of this study was to assess the utility of 24-hour composites as representative samples for measuring multiple microbiological targets in wastewater, and whether normalization of SARS-CoV-2 by endogenous targets can be used to decrease hour to hour variability at different watershed scales. Two sets of experiments were conducted, in tandem with the same wastewater, with samples collected at the building, cluster, and community sewershed scales. The first set of experiments focused on evaluating degradation of microbiological targets: SARS-CoV-2, Simian Immunodeficiency Virus (SIV) - a surrogate spiked into the wastewater, plus human waste indicators of Pepper Mild Mottle Virus (PMMoV), Beta-2 microglobulin (B2M), and fecal coliform bacteria (FC). The second focused on the variability of these targets from samples, collected each hour on the hour. Results show that SARS-CoV-2, PMMoV, and B2M were relatively stable, with minimal degradation over 24-h. SIV, which was spiked-in prior to analysis, degraded significantly and FC increased significantly over the course of 24 h, emphasizing the possibility for decay and growth within wastewater. Hour-to-hour variability of the source wastewater was large between each hour of sampling relative to the variability of the SARS-CoV-2 levels calculated between sewershed scales; thus, differences in SARS-CoV-2 hourly variability were not statistically significant between sewershed scales. Results further provided that the quantified representativeness of 24-h composite samples (i.e., statistical equivalency compared against hourly collected grabs) was dependent upon the molecular target measured. Overall, improvements made by normalization were minimal within this study. Degradation and multiplication for other targets should be evaluated when deciding upon whether to collect composite or grab samples in future studies.

Evaluation of per- and polyfluoroalkyl substances (PFAS) in leachate, gas condensate, stormwater and groundwater at landfills.

Per- and polyfluoroalkyl substances (PFAS), found in many consumer products, are commonly disposed of in landfills at the end of their service lives. To identify landfill liquids that should be prioritized for treatment, this study aimed to evaluate PFAS levels in different aqueous samples from landfills and identify relationships between PFAS and landfill characteristics. Twenty-six PFAS including 11 perfluoroalkyl carboxylic acids (PFCAs), 7 perfluoroalkyl sulfonates (PFSAs), and 8 perfluoroalkyl acid precursors (PFAA-precursors) were measured in municipal solid waste (MSW) leachate, construction and demolition debris (CDD) leachate, municipal solid waste incineration (MSWI) ash leachate, gas condensate, stormwater, and groundwater from landfills. Based on the median, results show that PFAS levels in MSW leachate were the highest (10,000 ng L-1), CDD leachate were intermediate (6200 ng L-1), and MSWI ash leachate were the lowest (1300 ng L-1) among the leachates evaluated. PFAS levels in gas condensate (7000 ng L-1) were similar to MSW leachate. PFAS in stormwater and groundwater were low (medians were less than 500 ng L-1). Dominant subgroups included PFCAs and PFAA-precursors in all leachates. PFSAs were also found in CDD leachate, PFAA-precursors in gas condensate, and PFCAs in stormwater and groundwater. Landfill characteristics significantly correlated with ∑26PFAS included waste proportions (percentage of MSWI ash in landfill, |rs| = 0.22), operational status (active or not, |rs| = 0.27) and rainfall (30-d cumulative rainfall, |rs| = 0.39). The results from this study can be used to prioritize which landfills and which reservoir of liquids (and corresponding subgroup of PFAS) to target for PFAS management.

Predicting COVID-19 cases using SARS-CoV-2 RNA in air, surface swab and wastewater samples.

Genomic footprints of pathogens shed by infected individuals can be traced in environmental samples, which can serve as a noninvasive method of infectious disease surveillance. The research evaluates the efficacy of environmental monitoring of SARS-CoV-2 RNA in air, surface swabs and wastewater to predict COVID-19 cases. Using a prospective experimental design, air, surface swabs, and wastewater samples were collected from a college dormitory housing roughly 500 students from March to May 2021 at the University of Miami, Coral Gables, FL. Students were randomly screened for COVID-19 during the study period. SARS-CoV-2 concentration in environmental samples was quantified using Volcano 2nd Generation-qPCR. Descriptive analyses were conducted to examine the associations between time-lagged SARS-CoV-2 in environmental samples and COVID-19 cases. SARS-CoV-2 was detected in air, surface swab and wastewater samples on 52 (63.4 %), 40 (50.0 %) and 57 (68.6 %) days, respectively. On 19 (24 %) of 78 days SARS-CoV-2 was detected in all three sample types. COVID-19 cases were reported on 11 days during the study period and SARS-CoV-2 was also detected two days before the case diagnosis on all 11 (100 %), 9 (81.8 %) and 8 (72.7 %) days in air, surface swab and wastewater samples, respectively. SARS-CoV-2 detection in environmental samples was an indicator of the presence of local COVID-19 cases and a 3-day lead indicator for a potential outbreak at the dormitory building scale. Proactive environmental surveillance of SARS-CoV-2 or other pathogens in multiple environmental media has potential to guide targeted measures to contain and/or mitigate infectious disease outbreaks within communities.

Expanding a Wastewater-Based Surveillance Methodology for DNA Isolation from a Workflow Optimized for SARS-CoV-2 RNA Quantification.

Wastewater-based surveillance (WBS) is a noninvasive, epidemiological strategy for assessing the spread of COVID-19 in communities. This strategy was based upon wastewater RNA measurements of the viral target, severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). The utility of WBS for assessing the spread of COVID-19 has motivated research to measure targets beyond SARS-CoV-2, including pathogens containing DNA. The objective of this study was to establish the necessary steps for isolating DNA from wastewater by modifying a long-standing RNA-specific extraction workflow optimized for SARS-CoV-2 detection. Modifications were made to the sample concentration process and included an evaluation of bead bashing prior to the extraction of either DNA or RNA. Results showed that bead bashing reduced detection of RNA from wastewater but improved recovery of DNA as assessed by quantitative polymerase chain reaction (qPCR). Bead bashing is therefore not recommended for the quantification of RNA viruses using qPCR. Whereas for Mycobacterium bacterial DNA isolation, bead bashing was necessary for improving qPCR quantification. Overall, we recommend 2 separate workflows, one for RNA viruses that does not include bead bashing and one for other microbes that use bead bashing for DNA isolation. The experimentation done here shows that current-standing WBS program methodologies optimized for SARS-CoV-2 need to be modified and reoptimized to allow for alternative pathogens to be readily detected and monitored, expanding its utility as a tool for public health assessment.

Relationships between SARS-CoV-2 in Wastewater and COVID-19 Clinical Cases and Hospitalizations, with and without Normalization against Indicators of Human Waste.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) in wastewater has been used to track community infections of coronavirus disease-2019 (COVID-19), providing critical information for public health interventions. Since levels in wastewater are dependent upon human inputs, we hypothesize that tracking infections can be improved by normalizing wastewater concentrations against indicators of human waste [Pepper Mild Mottle Virus (PMMoV), ß-2 Microglobulin (B2M), and fecal coliform]. In this study, we analyzed SARS-CoV-2 and indicators of human waste in wastewater from two sewersheds of different scales: a University campus and a wastewater treatment plant. Wastewater data were combined with complementary COVID-19 case tracking to evaluate the efficiency of wastewater surveillance for forecasting new COVID-19 cases and, for the larger scale, hospitalizations. Results show that the normalization of SARS-CoV-2 levels by PMMoV and B2M resulted in improved correlations with COVID-19 cases for campus data using volcano second generation (V2G)-qPCR chemistry (r s = 0.69 without normalization, r s = 0.73 with normalization). Mixed results were obtained for normalization by PMMoV for samples collected at the community scale. Overall benefits from normalizing with measures of human waste depend upon qPCR chemistry and improves with smaller sewershed scale. We recommend further studies that evaluate the efficacy of additional normalization targets.