Wastewater Treatment Plant Samples Research Articles

Assessing environmental exposure to viruses in wastewater treatment plant and swine farm scenarios with next-generation sequencing and occupational risk approaches

Occupational exposure to pathogens can pose health risks. This study investigates the viral exposure of workers in a wastewater treatment plant (WWTP) and a swine farm by analyzing aerosol and surfaces samples. Viral contamination was evaluated using quantitative polymerase chain reaction (qPCR) assays, and target enrichment sequencing (TES) was performed to identify the vertebrate viruses to which workers might be exposed. Additionally, Quantitative Microbial Risk Assessment (QMRA) was conducted to estimate the occupational risk associated with viral exposure for WWTP workers, choosing Human Adenovirus (HAdV) as the reference pathogen. In the swine farm, QMRA was performed as an extrapolation, considering a hypothetical zoonotic virus with characteristics similar to Porcine Adenovirus (PAdV). The modelled exposure routes included aerosol inhalation and oral ingestion through contaminated surfaces and hand-to-mouth contact.HAdV and PAdV were widespread viruses in the WWTP and the swine farm, respectively, by qPCR assays. TES identified human and other vertebrate viruses WWTP samples, including viruses from families such as Adenoviridae, Circoviridae, Orthoherpesviridae, Papillomaviridae, and Parvoviridae. In the swine farm, most of the identified vertebrate viruses were porcine viruses belonging to Adenoviridae, Astroviridae, Circoviridae, Herpesviridae, Papillomaviridae, Parvoviridae, Picornaviridae, and Retroviridae.QMRA analysis revealed noteworthy risks of viral infections for WWTP workers if safety measures are not taken. The probability of illness due to HAdV inhalation was higher in summer compared to winter, while the greatest risk from oral ingestion was observed in workspaces during winter. Swine farm QMRA simulation suggested a potential occupational risk in the case of exposure to a hypothetical zoonotic virus.This study provides valuable insights into WWTP and swine farm worker's occupational exposure to human and other vertebrate viruses. QMRA and NGS analyses conducted in this study will assist managers in making evidence-based decisions, facilitating the implementation of protection measures, and risk mitigation practices for workers.

Measuring SARS-CoV-2 RNA concentrations in neighborhood wastewater

Longitudinal wastewater sampling during the COVID-19 pandemic was an important aspect of disease surveillance, adding to a more complete understanding of infection dynamics and providing important data for community public health monitoring and intervention planning. This was largely accomplished by testing SARS-CoV-2 RNA concentrations in samples from municipal wastewater treatment plants (WWTPs). We evaluated the utility of testing for virus levels upstream from WWTP within the residential neighborhoods that feed into the WWTP. We propose that monitoring virus dynamics across residential neighborhoods could reveal important public health-relevant information about community sub-group heterogeneity in virus concentrations. Principal resultsVirus concentration patterns display heterogeneity within neighborhoods and between neighborhoods over time. Sewage SARS-CoV-2 RNA concentrations as measured by RT-qPCR also corresponded closely to verified COVID-19 infection counts within individual neighborhoods. More importantly, our data suggest the loss of disease-relevant public health information when sampling occurs only at the level of WWTP instead of upstream in neighborhoods. Spikes in SARS-CoV-2 RNA concentrations in neighborhoods are often masked by dilution from other neighborhoods in the WWTP samples. Major conclusionsWastewater-based epidemiology (WBE) employed at WWTP reliably detects SARS-CoV-2 in a city-sized population but provides less actionable public health information about neighborhoods experiencing greater viral infection and disease. Neighborhood sewershed sampling reveals important population-based information about local virus dynamics and improves opportunities for public health intervention. Longitudinally employed, neighborhood sewershed surveillance may provide a 3–6 day early warning of SARS-CoV-2 infection spikes and, importantly, highly specific information on subpopulations in a community particularly at higher risk at different points in time. Sampling in neighborhoods may thus provide timely and cost-saving information for targeted interventions within communities.

Global abundance patterns, diversity, and ecology of Patescibacteria in wastewater treatment plants

BackgroundMicroorganisms are responsible for nutrient removal and resource recovery in wastewater treatment plants (WWTPs), and their diversity is often studied by 16S rRNA gene amplicon sequencing. However, this approach underestimates the abundance and diversity of Patescibacteria due to the low coverage of commonly used PCR primers for this highly divergent bacterial phylum. Therefore, our current understanding of the global diversity, distribution, and ecological role of Patescibacteria in WWTPs is very incomplete. This is particularly relevant as Patescibacteria are considered to be associated with microbial host cells and can therefore influence the abundance and temporal variability of other microbial groups that are important for WWTP functioning.ResultsHere, we evaluated the in silico coverage of widely used 16S rRNA gene-targeted primer pairs and redesigned a primer pair targeting the V4 region of bacterial and archaeal 16S rRNA genes to expand its coverage for Patescibacteria. We then experimentally evaluated and compared the performance of the original and modified V4-targeted primers on 565 WWTP samples from the MiDAS global sample collection. Using the modified primer pair, the percentage of ASVs classified as Patescibacteria increased from 5.9 to 23.8%, and the number of detected patescibacterial genera increased from 560 to 1576, while the detected diversity of the remaining microbial community remained similar. Due to this significantly improved coverage of Patescibacteria, we identified 23 core genera of Patescibacteria in WWTPs and described the global distribution pattern of these unusual microbes in these systems. Finally, correlation network analysis revealed potential host organisms that might be associated with Patescibacteria in WWTPs. Interestingly, strong indications were found for an association between Patescibacteria of the Saccharimonadia and globally abundant polyphosphate-accumulating organisms of the genus Ca. Phosphoribacter.ConclusionsOur study (i) provides an improved 16S rRNA gene V4 region-targeted amplicon primer pair inclusive of Patescibacteria with little impact on the detection of other taxa, (ii) reveals the diversity and distribution patterns of Patescibacteria in WWTPs on a global scale, and (iii) provides new insights into the ecological role and potential hosts of Patescibacteria in WWTPs.9DpTXjuT--dysnV-FwKdBeVideo

Wastewater-based epidemiology applied at the building-level reveals distinct virome profiles based on the age of the contributing individuals

BackgroundHuman viruses released into the environment can be detected and characterized in wastewater. The study of wastewater virome offers a consolidated perspective on the circulation of viruses within a population. Because the occurrence and severity of viral infections can vary across a person’s lifetime, studying the virome in wastewater samples contributed by various demographic segments can provide valuable insights into the prevalence of viral infections within these segments. In our study, targeted enrichment sequencing was employed to characterize the human virome in wastewater at a building-level scale. This was accomplished through passive sampling of wastewater in schools, university settings, and nursing homes in two cities in Catalonia. Additionally, sewage from a large urban wastewater treatment plant was analysed to serve as a reference for examining the collective excreted human virome.ResultsThe virome obtained from influent wastewater treatment plant samples showcased the combined viral presence from individuals of varying ages, with astroviruses and human bocaviruses being the most prevalent, followed by human adenoviruses, polyomaviruses, and papillomaviruses. Significant variations in the viral profiles were observed among the different types of buildings studied. Mamastrovirus 1 was predominant in school samples, salivirus and human polyomaviruses JC and BK in the university settings while nursing homes showed a more balanced distribution of viral families presenting papillomavirus and picornaviruses and, interestingly, some viruses linked to immunosuppression.ConclusionsThis study shows the utility of building-level wastewater-based epidemiology as an effective tool for monitoring the presence of viruses circulating within specific age groups. It provides valuable insights for public health monitoring and epidemiological studies.

Development and application of the UVC LED-based spectrophotometer for high frequency online monitoring of nitrate concentration in municipal wastewater

Developing a fast, sensitive and stable sensor for online monitoring nitrate concentration is an urgent need for the optimization of denitrification processes in wastewater treatment plants (WWTPs). In this study, the miniaturized spectrophotometric nitrate sensor was developed using UVC light-emitting diodes (LEDs) coupled with AlGaN or SiC-based photodiodes. The use of novel SiC photodiodes could effectively eliminate UVB and UVA light in the 230 nm LED emission spectra. Replacing 235 nm UVC-LED with 230 nm UVC-LED could effectively increase the detection sensitivity but not the linear range that follows the Lambert-Beer's law. The influences of dissolved organic matter (DOM) on nitrate analysis could be corrected by a unified coefficient α across the NO3--N concentration ranging in 0–50 mg/L. Across the investigated WWTP samples, the correction coefficient α was mainly determined by the fractions of humic substances with apparent molecular weight (MW) of 1.5–9.0 kDa, while the degradation of the large MW proteinaceous biopolymer resulted in dramatic variations of correction coefficient α along the WWTP A/A/O process. Microfiltration membranes with pore sizes ≤ 30 µm could sufficiently eliminate the influences of particles in effluents on photometric nitrate analysis. The ultrafiltration and enhanced coagulation treatment could effectively remove the biopolymer fractions of DOM in wastewater, and thus alleviate its influences on sensitivity and accuracy of nitrate analysis. The spectrophotometric nitrate sensors were successfully applied for on-line monitoring NO3--N concentrations in the influent and effluent of denitrifying biofilter in a municipal WWTP, providing high-frequency feedback signals to precise control of feeding external carbon source.

Occurrence, behavior and fate of liquid crystal monomers in municipal wastewater

Liquid crystal monomers (LCMs), the essential substances used in the display screen of electronic devices, have been proposed as a class of emerging chemicals of concern. Despite their detection in various environmental matrices, little is known about the presence of LCMs in municipal sewage systems. This study aimed to investigate the occurrence, distribution, and fate of 64 LCMs released into the aqueous environment from a municipal wastewater treatment plant (WWTP) in Hong Kong, China. In total 14 LCMs were detected in WWTP samples. Specifically, the Σ14LCMs concentrations in crude influent, final effluent, and final sludge were found to be 16.8 ± 0.3 ng/L, 2.71 ± 0.05 ng/L, and 19.2 ± 1.0 ng/g dry weight, respectively. Among them, 10 fluorinated LCMs (F-LCMs) were determined to be present at concentrations of 8.90 ± 0.10 ng/L, 1.69 ± 0.05 ng/L, and 9.94 ± 1.00 ng/g dry weight, respectively. The predominant non-fluorinated LCMs (NF-LCMs) detected in all samples were 3OCB and EPhEMOB, while 2OdF3B was the dominant F-LCM. The overall removal rate of total LCMs was 83.8 ± 0.3 %, with 25.4 ± 4.8 % being removed by biodegradation and UV treatment. Compared to NF-LCMs, F-LCMs were more resistant to biodegradation. Despite the significant removal of LCMs through WWTP, the remaining LCMs in final effluent could result in an annual emission of 3.04 kg of total LCMs from the population of Hong Kong. This study provides the first evidence of LCMs contamination in municipal wastewater, possibly arising from routine electronic devices usage. Further investigation is needed to elucidate the potential impact of LCMs emission via WWTP effluent on the aquatic receiving ecosystem.

Assessing eco-efficiency of wastewater treatment plants: A cross-evaluation strategy

Evaluating the eco-efficiency of wastewater treatment plants (WWTPs) is crucial for enhancing environmental and economic performance in the water utility sector. Previous studies in this area estimated WWTP eco-efficiency through self-evaluation, which might have led to overestimation and biased policy recommendations. To address these issues, this study applies a cross-evaluation strategy, combining self-evaluation and peer-evaluation, to assess the eco-efficiency of WWTPs. The empirical application focuses on a sample of Spanish WWTPs, yielding the following key findings. Average eco-efficiency scores were 0.353 and 0.230, for self-evaluation and global peer-evaluation approaches, respectively, confirming the overestimation of eco-efficiency scores based on self-evaluation. If WWTPs were eco-efficient, they could potentially reduce greenhouse gas (GHG) emissions by up to 0.39 kg CO2eq/year. The application of reliable methods, such as peer-evaluation, for eco-efficiency assessment of WWTPs provides water regulators with a comprehensive understanding of the environmental and economic performance of WWTPs. This knowledge guides decision-making, policy development, and resource allocation, facilitating sustainable and efficient wastewater management practices.

Dissemination of carbapenemase-producing Enterobacterales through wastewater and gulls at a wastewater treatment plant in Sweden

Here we report the detection of carbapenemase-producing Enterobacterales (CPE) isolated from Swedish wastewater and gull faeces. CPE have not been detected in samples from animals in Sweden preceding this report. Sampling of wastewater treatment plant (WWTP) inlet and outlet, sedimentation basins, surface seawater from key aquatic bird habitats and freshly deposited gull faeces was done on six separate occasions during May to September 2021. Following broth enrichment, selective screening of putative CPE was performed on mSuperCarba™ (CHROMagar). Species identification was done with MALDI-TOF. Antimicrobial susceptibility testing was performed according to EUCAST. In total, seventeen CPE were verified by genome sequencing carrying blaGES-5, blaIMI-3, blaOXA-181 or blaOXA-244. The blaGES-5 was carried on IncP plasmids in four different species; Escherichia coli ST10 isolated from WWTP outlet, Raoultella ornithinolytica isolated from WWTP inlet, outlet and sedimentation basins as well as gull faeces collected at the WWTP and Klebsiella spp. isolates from WWTP inlet and outlet. The genetic environment surrounding blaGES-5 was similar in two Citrobacter freundii causing human infections. The blaIMI-3 was carried on IncFII(Yp) plasmids in four Enterobacter ludwigii, isolated from WWTP outlet and gull faeces collected at a recreational city park 2 km from the WWTP. The blaOXA-181 was located on a COLKP3 plasmid found in an E. coli, while blaOXA-244 was chromosomally located in an E. coli ST10, both isolated from WWTP inlet. Phylogenetic analysis of R. ornithinolytica and E. ludwigii isolates indicate that the gulls carried strains related to those identified in the WWTP samples. The results thus add to the increasing evidence of WWTPs as anthropogenic reservoirs for mobile genetic elements with antibiotic-resistance functionality. Such environments could profoundly impact the dissemination and spread of such genetic elements via for example aquatic birds, thereby warranting further study and surveillance.

Detection of Retinoic Acid-Active Chemicals in Diverse Sample Matrices Via a Quantum Dots-Based Nuclear Receptor Fluorescence Probe-Mediated Biosensor.

Developing a sensitive and reliable method for the screening of various endocrine-disrupting chemicals (EDCs) is in high demand and yet remains a significant challenge. Herein, we developed a CdSe/ZnS QDs-based nuclear receptor fluorescence probe (QDs-NRFP)-mediated biosensor for the screening of retinoic acid (RA)-active chemicals (a class of EDCs). The QDs-NRFP can be prepared on the spot via an antigen-antibody immunobinding interaction between the GST tag of the human retinoic acid receptor β ligand-binding domain (GST-hRARβ-LBD) and the CdSe/ZnS QDs-labeled anti-GST tag antibody. It can not only maintain the high binding activity of GST-hRARβ-LBD but also improve the sensitivity due to the high quantum yield of CdSe/ZnS QDs. Based on the indirect competition bioassay, the developed biosensor showed a detection limit of 1.8 ng/L all-trans-retinoic acid binding activity equivalent (atRA-BAE) with a linear range of 7.5-1183.6 ng/L. Compared with many cell-dependent in vitro assays, the QDs-NRFP-mediated biosensor is cell-free and unaffected by the cytotoxic substances in matrices and exhibited obvious superiority in detection time (within 40 min) and accuracy. As a case study, the biosensor was applied to detect RA binding activities in various sample matrices obtained from a wastewater treatment plant (WWTP) and physiological samples and showed satisfactory accuracy and reliability. The developed QDs-NRFP-mediated biosensor is expected to be capable of screening various EDCs with universality based on different nuclear receptor signaling pathways, which will substantially accelerate the assessment of global EDCs.

Exploring the correlations between epi indicators of COVID-19 and the concentration of pharmaceutical compounds in wastewater treatment plants in Northern Portugal

The COVID-19 pandemic caused by the SARS-CoV-2 virus led to changes in the lifestyle and human behaviour, which resulted in different consumption patterns of some classes of pharmaceuticals including curative, symptom-relieving, and psychotropic drugs. The trends in the consumption of these compounds are related to their concentrations in wastewater systems, since incompletely metabolised drugs (or their metabolites back transformed into the parental form) may be detected and quantified by analytical methods.Pharmaceuticals are highly recalcitrant compounds and conventional activated sludge processes implemented in wastewater treatment plants (WWTP) are ineffective at degrading these substances. As a results, these compounds end up in waterways or accumulate in the sludge, being a serious concern given their potential effects on ecosystems and public health. Therefore, it is crucial to evaluate the presence of pharmaceuticals in water and sludge to assist in the search for more effective processes. In this work, eight pharmaceuticals from five therapeutic classes were analysed in wastewater and sludge samples collected in two WWTP located in the Northern Portugal, during the third COVID-19 epidemic wave in Portugal. The two WWTP demonstrated a similar pattern with respect to the concentration levels in that period. However, the drugs loads reaching each WWTP were dissimilar when normalising the concentrations to the inlet flow rate. Acetaminophen (ACET) was the compound detected at highest concentrations in aqueous samples of both WWTP (98. 516 μg L − 1 in WWTP2 and 123. 506 μg L − 1in WWTP1), indicating that this drug is extensively used without the need of a prescription, known of general public knowledge as an antipyretic and analgesic agent to treat pain and fever. The concentrations determined in the sludge samples were below 1.65 µg g − 1 in both WWTP, the highest value being found for azithromycin (AZT). This result may be justified by the physico-chemical characteristics of the compound that favour its adsorption to the sludge surface through ionic interactions. It was not possible to establish a clear relationship between the incidence of COVID-19 cases in the sewer catchment and the concentration of drugs detected in the same period. However, looking at the data obtained, the high incidence of COVID-19 in January 2021 is in line with the high concentration of drugs detected in the aqueous and sludge samples but prediction of drug load from viral load data was unfeasible.

Dispersive Liquid - Liquid Micro extraction Technique Coupled with High Performance Liquid Chromatography Diode Array Detector for the Determination of Sulfonamides in Water Samples

A dispersive liquid-liquid microextraction combined with high-performance liquid chromatography with diode array detection (DLLME-HPLC-DAD) has been developed for the determination of 15 sulfonamides in water samples. The effects of experimental parameters on the performance of the method such as the type and volume of extraction and disperser solvents, pH of the solution, effect of salt, and centrifugation time were evaluated, and optimum conditions were established. Under these optimum conditions, linearity was found in the range of 2 - 1 000 μg L-1 with regression coefficients better than 0.9990. The limit of detection (LOD) and limit of quantification (LOQ) values were in the range of 0.6 - 7.8 μg L-1 and 1.5 - 8.3 µg L-1, respectively. Intra-day and inter-day precision ranged from 1.5 - 9.7% and from 3.1 - 9.9%, respectively. The accuracy of the method were also evaluated in samples from three different sources of water (river water, groundwater, and tap water) were found to range from 70.5 to 103.9% with %RSD values ranging from 0.5 to 9.8%, with the exception of sulfaguanidine in river water and groundwater where the percentage recovery ranged from 54.3 - 84.9% at the two higher concentration levels (100 and 400 µg L-1).Furthermore, the effect of sample matrix on the extraction of target compounds was evaluated using the relative recovery (%RR) of the samples from three different water sources relative to the ultrahigh purity (UHP) water sample spiked with target analytes, and found in the range of 70.3 - 106.1%.The proposed method was successfully applied to wastewater treatment plant samples and Sulfisoxazole (SSO) was detected in the influent samples in the range of 14.8 - 17.8 µg L-1 with corresponding %RSD, (n = 5) value of 1.6% to 1.9%.The results indicated that the proposed method is effective for the extraction and determination of the sulfonamides in water samples.

Comprehensive micropollutant characterization of wastewater during Covid-19 crisis in 2020: Suspect screening and environmental risk prioritization strategy

Micropollutants monitoring in wastewater can serve as a picture of what is consuming society and how it can impact the aquatic environment. In this work, a suspect screening approach was used to detect the known and unknown contaminants in wastewater samples collected from two wastewater treatment plants (WWTPs) located in the Basque Country (Crispijana in Alava, and Galindo in Vizcaya) during two weekly sampling campaigns, which included the months from April to July 2020, part of the confinement period caused by COVID-19. To that aim, high-resolution mass spectrometry was used to collect full-scan data-dependent tandem mass spectra from the water samples using a suspect database containing >40,000 chemical substances. The presence of > 80 contaminants was confirmed (level 1) and quantified in both WWTP samples, while at least 47 compounds were tentatively identified (2a). Among the contaminants of concern, an increase in the occurrence of some compounds used for COVID-19 disease treatment, such as lopinavir and hydroxychloroquine, was observed during the lockdown. A prioritization strategy for environmental risk assessment was carried out considering only the compounds quantified in the effluents of Crispijana and Galindo WWTPs. The compounds were scored based on the removal efficiency, estimated persistency, bioconcentration factor, mobility, toxicity potential and frequency of detection in the samples. With this approach, 33 compounds (e.g. amantadine, clozapine or lopinavir) were found to be considered key contaminants in the analyzed samples based on their concentration, occurrence and potential toxicity. Additionally, antimicrobial (RQ-AR) and antiviral (EDRP) risk of certain compounds was evaluated, where ciprofloxacin and fluconazole represented medium risk for antibiotic resistance (1 > RQ-AR > 0.1) in the aquatic ecosystems. Regarding mixture toxicity, the computed sum of toxic unit values of the different effluents (> 1) suggest that interactions between the compounds need to be considered for future environmental risk assessments.

Are the ecotoxicological tools viable to evaluate the effectiveness of wastewater treatment plant effluents?

Ecotoxicological tools have proved to be sensitive and appropriate for the evaluation of the effectiveness of treatments used in wastewater treatment plants (WWTP). The objective of this study was to assess the applicability of bioassays and biomarkers to evaluate the efficiency of different treatments throughout WWTP samples[A—raw influent, B—preliminary effluent, C—final effluent, and D—receiving stream], seasonally over 1 year, through a multispecies approach: i) bacterial cell viability [Escherichia coli, Rhodopirellulla rubra, Arthrobacter sp., and Pseudomonas putida]; ii) microalgae Raphidocelis subcapitata and the macrophyte Lemna minor growth inhibition; and iii) microcrustacean Daphnia magna acute and feeding rate assays. Total chlorophyll, malondialdehyde, and proline levels were evaluated in L. minor, and catalase, glutathione-S-transferase activities, and thiobarbituric acid reactive substances levels were quantified in D. magna, after exposure to wastewater samples. Overall, the tested species showed different sensitivities, P. putida = Arthrobacter sp. = R. rubra < R. subcapitata < E. coli = D. magna = L. minor, to the collected samples. The results obtained in D. magna and L. minor assays demonstrated that these organisms can be used in programs for monitoring and environmental assessment of wastewater effluents. The present study demonstrates the usefulness of ecotoxicological tools, with multispecies and different endpoints, to assess the effectiveness of WWTPs. Moreover, it is important to ensure that WWTP implements a monitoring program to minimize the discharge of effluents that compromise the environment in order to guarantee the good ecological quality of the environmental ecosystems.

Biotic transformation products of sulfonamides in environmental water samples: High-resolution mass spectrometry-based tentative identification by a suspect screening approach

The presence of pharmaceuticals in the aquatic environment is mainly due to their release from the effluents of the wastewater treatment plants (WWTPs), which are unable to completely remove them and their transformation products (TPs). Sulfonamides (SAs) are a synthetic antibacterial class used for the treatment of both human and animal infections; they have often been reported in surface water, thus contributing to the antibiotic resistance emergency. Monitoring SA TPs should be important as well because they could still exert some pharmaceutical activity; however, many TPs are still unknown since several transformation processes are possible (e. g. human and animal metabolism, WWTP activities, environmental factors etc.). In this work, three of the most used SAs, i.e., sulfamethoxazole (SMX), sulfapyridine (SPY), and sulfadiazine (SDZ), were incubated for 20 days in a batch reactor with activated sludge under controlled conditions. Then, the water sample was extracted and analyzed by ultra-high performance liquid chromatography-high resolution mass spectrometry in the data dependent acquisition (DDA) mode. Starting from the literature data, the possible transformation pathways were studied, and for each SA, a list of TPs was hypothesized and used for the identification. The raw data files were processed with Compound Discoverer, and 44 TPs (18, 13, and 13 TPs for SMX, SPY, and SDZ, respectively), including multiple TPs, were manually validated. To overcome the limitation of the DDA, the identified TPs were used in an inclusion list to analyze WWTP samples by a suspect screening approach. In this way, 4 SMX TPs and 5 SPY TPs were tentatively identified together with their parent compounds. Among these TPs, 5 of 9 were acetylated forms, in agreement with previous literature reporting that acetylation is the predominant SA transformation.

Wastewater surveillance of SARS-CoV-2 and influenza in preK-12 schools shows school, community, and citywide infections

Wastewater surveillance is a passive and efficient way to monitor the spread of infectious diseases in large populations and high transmission areas such as preK-12 schools. Infections caused by respiratory viruses in school-aged children are likely underreported, particularly because many children may be asymptomatic or mildly symptomatic. Wastewater monitoring of SARS-CoV-2 has been studied extensively and primarily by sampling at centralized wastewater treatment plants, and there are limited studies on SARS-CoV-2 in preK-12 school wastewater. Similarly, wastewater detections of influenza have only been reported in wastewater treatment plant and university manhole samples. Here, we present the results of a 17-month wastewater monitoring program for SARS-CoV-2 (n = 2176 samples) and influenza A and B (n = 1217 samples) in 51 preK-12 schools. We show that school wastewater concentrations of SARS-CoV-2 RNA were strongly associated with COVID-19 cases in schools and community positivity rates, and that influenza detections in school wastewater were significantly associated with citywide influenza diagnosis rates. Results were communicated back to schools and local communities to enable mitigation strategies to stop the spread, and direct resources such as testing and vaccination clinics. This study demonstrates that school wastewater surveillance is reflective of local infections at several population levels and plays a crucial role in the detection and mitigation of outbreaks.

New insight into fate and transport of organic compounds from pollution sources to aquatic environment using non-targeted screening: A wastewater treatment plant case study

A variety of chemicals discharged into the aquatic environment by the wastewater treatment plant (WWTP), which is a potential source of hazard to the ecological environment and human health. This study established a novel analytical method for all compounds using non-targeted screening to comprehensively explore the fate and transport of organic compounds from WWTP to aquatic environment. 3967 and 3636 features were detected in WWTP samples and river samples, respectively. Multi-level classification was applied to all identified compounds, and results showed that aliphatics were dominant in both abundance and response, accounting for an average of 35.49 % and 74.10 %, respectively. A total of 88 Emerging Contaminants (ECs), including 22 endocrine disrupting chemicals (EDCs), 12 pharmaceuticals and personal care products (PPCPs), 12 pesticides, 10 volatile organic compounds (VOCs), 5 persistent organic pollutants (POPs) and 27 chemicals with other uses, were identified from all compounds, and their traceability analysis was performed. Furthermore, the contribution rate of organic compounds from WWTP effluent to river was calculated to be 33.60 % by the analysis of source-sink relationship. An in-depth and comprehensive exploration of the fate and transport of all organic compounds will help to provide guidelines for the treatment technologies and achieve the traceability of pollutants.

1874. Comprehensive, longitudinal wastewater surveillance for SARS-CoV-2 across a university campus, demonstrates low levels of SARS-CoV-2 activity relative to the surrounding community

Abstract Background Universities are interactive communities where frequent contacts between individuals occur, increasing the risk of outbreaks of COVID-19. We embarked upon a real-time wastewater (WW) monitoring program across the University of Calgary (UofC) campus measuring WW SARS-CoV-2 burden relative to levels of disease in the broader surrounding community. Figure 1 The colour scheme shows 6 sewer sub-catchments at the University of Calgary. Autosamplers were deployed at 4 sampling nodes within sub-catchments CR and YA (both residence halls), and UCE and UCS (catchments that include several campus buildings). Figure 2 Log10-transformed abundance (i.e., copies per mL) of nucleocapsid gene (i.e., N1) for SARS-CoV-2 for each sampling location during October 2021 – April 2022. Locations denoted by the same letters (A, B, or C) show no statistical difference (p &amp;gt; 0.05) according to the Wilcoxon rank-sum test. The WWTP sample corresponds to a catchment area covering most of Calgary including the university campus, for which sampling locations CR, UCE, UCS, and UCW are defined in Fig. 1. Methods From October 2021 – April 2022, WW was collected thrice weekly across UofC campus through 4 individual sewer sampling nodes (Fig. 1) using autosamplers (C.E.C. Analytics, CA). Results from these 4 nodes were compared with community monitoring at Calgary’s largest WW treatment plant (WWTP), which received WW from surrounding neighborhoods, and also from UofC. Nucleic acid was extracted from WW for RTqPCR quantification of the N1 nucleocapside gene from SARS-CoV-2 genomic RNA. Qualitative (positive samples defined if cycle threshold &amp;lt; 40) and quantitative statistical analyses were performed using R. Results Levels of SARS-CoV-2 in WW were significantly lower at all campus monitoring sites relative to the WWTP (Wilcoxon rank-sum test p &amp;lt; 0.05; Fig. 2). The proportion of WW samples that were positive for SARS-CoV-2 was significantly higher for WWTP than at least two campus locations (p &amp;lt; 0.05 for Crowsnest Hall and UCE - University way and campus drive) according to Fischer’s exact 2-sided test. The proportion of WW samples with positive WW signals were still higher for WWTP than the other two locations, but statistically not significant (p = 0.216). Among campus locations, the buildings in UCE catchment showed much lower N1 signals than other catchments, likely owing to buildings in this catchment primarily being administration and classroom environments, with lower human-to-human contact and less defecation compared to the other 3 catchments, which include residence hall, a dining area, and/or laboratory spaces. Conclusion Our results show that SARS-CoV-2 RNA shedding in WW at the UofC is significantly lower than the city-wide signal associated with surrounding neighborhoods. Furthermore, we demonstrate that WW testing at well-defined nodes is a sampling strategy for potentially locating specific places where high transmission of infectious disease occurs. Disclosures All Authors: No reported disclosures.

Mastadenovirus Molecular Diversity in Waste and Environmental Waters from the Lisbon Metropolitan Area.

In face of the absence of epidemiological data regarding the circulation of human adenoviruses (HAdV) in Portugal, this study aimed at the evaluation of their molecular diversity in waste and environmental waters in the Lisbon Metropolitan Area (LMA). Using samples collected between 2018 and 2021, the HAdV hexon protein-coding sequence was partially amplified using three nested touch-down PCR protocols. The amplification products obtained were analyzed in parallel by two approaches: molecular cloning followed by Sanger sequencing and Next-Generation Sequencing (NGS) using Illumina® sequencing. The analysis of NGS-generated data allowed the identification of a higher diversity of HAdV-A (19%), -B (1%), -C (3%), -D (24%), and -F (25%) viral types, along with murine adenovirus (MAdV-2; 30%) in the wastewater treatment plant samples. On the other hand, HAdV-A (19%), -D (32%), and -F (36%) were identified in environmental samples, and possibly MAdV-2 (14%). These results demonstrate the presence of fecal contamination in environmental waters and the assessment of the diversity of this virus provides important information regarding the distribution of HAdV in LMA, including the detection of HAdV-F41, the most frequently reported in water worldwide.

Prevalence of Multiple Drug-Resistant Bacteria in the Main Campus Wastewater Treatment Plant of Wolaita Sodo University, Southern Ethiopia.

Wastewater treatment plants (WWTPs) are important reservoirs for the development of drug resistance and a potential route for the dissemination of antibiotic resistance genes (ARGs) in the environment. One of the most serious challenges in Ethiopia is the widespread emergence of antibiotic resistance among bacterial pathogens. The bacteria were isolated between September 2018 and May 2019 from the main campus of Wolaita Sodo University in Southern Ethiopia. Using an enrichment process and selective media isolation, 380 wastewater treatment plant samples were collected and screened for the presence of various bacterial isolates. Of a total of 380 wastewater treatment samples, 136 were isolated. Positive prevalence was documented in 136 sample isolates of bacteria from six genera. Escherichia coli 34 (8.94%), Salmonella spp. 15 (3.94%), Shigella spp. 32 (8.42%), Staphylococcus aureus 23 (6.05%), Pseudomonas aeruginosa 21 (5.52%), and Proteus spp. 11 (2.89%). The general prevalence of bacterial isolates was assessed, and 136 (37.58%) samples tested positive for culture. Furthermore, isolates were used to determine sensitivity/resistance patterns using the Kirby-Bauer disc diffusion method and the agar well diffusion technique, respectively. Multiple drug resistance isolates and multiple values of the antibiotic resistance index were evaluated and recorded according to the resistant pattern. Some organisms were sensitive to sparfloxacin and tobramycin, while Staphylococcus aureus was sensitive to methicillin and others showed the highest resistance. At least four of the seven antibiotic classes were found to be resistant to multiple drug resistance isolates, and some classes of antibiotics were found to be highly sensitive to these isolates. Multiple antibiotic resistance index values ranged from 0.37 to 0.75, with Salmonella spp., Shigella spp., and Staphylococcus aureus having the highest score values. The current study has shown that some of the bacterial isolates were resistant to common antibiotics. Therefore, it is recommended that the emergence of multiple drug resistance increased rapidly, pathogenic bacteria inappropriate treated wastewater treatment plant systems were continuously contaminated, and bacterial resistance increased day by day as a result of environmental factors. As a result, due to the serious challenges facing the community's health, multiple drug-resistant prevention and control strategies must be implemented.

An advanced LC-MS/MS protocol for simultaneous detection of pharmaceuticals and personal care products in the environment.

The development of appropriate analytical screening techniques for pharmaceuticals and personal care products (PPCPs) is the basis for studying the distribution and environmental impact of emerging contaminants (ECs). Mass spectrometry-based screening methods vary with the complexity of the target compounds. It is challenging to balanceboth positive and negative ion quantification with a low detection limit. To establish a set of experimental methods including extraction, chromatography-separation and mass spectrometry screening is one of the most important topics in PPCP research. This paper describes a universal and efficient qualification and quantification protocol for the simultaneous detection of 34 PPCPs in different environmental samples in a single analytical data acquisition run. Thirty-four representative PPCPs, which are widely distributed in the environment with high ecological toxicity and complex chemical structures, were selected as representative target ECs. The extraction of the target PPCPs was achieved using only one solid-phase extraction cartridge without the need to adjust the pH of samples. The enriched samples were detected by LC-MS/MS in both positive and negative ion modes simultaneously. The protocol was evaluated based on the accuracy, precision, detection limits and matrix effects. This method achieved simultaneous detection of PPCPs in both positive and negative ion modes, with a single analytical cycle of 12 min. The observed SPE recoveries were between 40% and 115%. The instrumental detection limits (IDL) varied from 0.01 to 1 pg, and the method detection limits (MDL) were between 0.002 and 3.323 ng/l in different matrices. Most of the PPCPs were subjected to matrix suppression below 30%. The method was successfully applied for quantitative analysis of the PPCPs in different environmental samples, including river samples, wastewater treatment plant (WWTP) samples and soil samples. This protocol developed a rapid and efficient detection method to simultaneous qualitative and quantitative 34 representative PPCPs in the environment. The IDL ranged from 0.01 to 1 pg and the MDL ranged from 0.002 to 3.323 ng/l in different matrices. The detection limit was one order of magnitude lower compared to previous studies. The protocol also provided a wide application range for different environmental matrices, which permitted the migration and transformation of PPCPs to be explored.