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In this work, a rapid and simplified method for extracting SARS-CoV-2 RNA from whole wastewater using a magnetic beads-based protocol is presented. The described method involves the centrifugation of a 50-mL aliquot of raw wastewater influent for 5 min to obtain a 500-μL pellet, which is eluted with 2 mL of a Tween®20-based elution buffer; 1 mL of the elute is extracted for RNA using a direct magnetic bead-based extraction method. RNA recovery was examined in several bench-scale experiments using heat-inactivated SARS-CoV-2 (HI-SCV-2) spiked into raw wastewater to assess the effects of different solids pellet : buffer ratios, inhibition mitigation strategies, and varying levels of total suspended solids. When the method was assessed using an influent wastewater sample known to contain SARS-CoV-2, the viral signal was detected in all five biological replicates, whereas direct extraction of 1-mL aliquots of the raw wastewater resulted in a 40% viral detection rate. The experimental method limit of detection (MLOD) using HI-SCV-2 spiked into raw wastewater was 50 GU mL−1 with a 95% limit of detection. Using the described protocol, the presence of SARS-CoV-2 RNA was verified in wastewater collected from wastewater treatment facilities (WWTFs) in Atlantic Canada over a period of 15 weeks during the rise and fall of a COVID-19 outbreak. This method is effective and rapid and could provide potential application for laboratories with limited resources. Of approximately 50 methods that have been developed for measuring SARS-CoV-2 in wastewater referenced in the literature, this is the first to advance a robust magnetic beads-based RNA extraction technique from whole wastewater without extensive sample pre-treatment. The novel application of this method in the rapid extraction of SARS-CoV-2 RNA from municipal wastewater is an indispensable tool to potentially understand COVID-19 infection occurrence within communities.

The overall objective of this work was to develop a simple and effective passive sampling protocol for the detection of SARS-CoV-2 in sewer catchments at targeted institutional-level sampling sites in a region of low COVID-19 prevalence. We developed a new 3D-printed sampling cage and assessed four commercially-available materials (cotton gauze, cotton cheesecloth, cellulose sponges, and electronegative filters) for RNA adsorption in the cage. We determined that cheesecloth and electronegative filters provided an effective approach for collecting and measuring SARS-CoV-2 in wastewater. We also compared the performance of three elution mixtures (a commercially-available lysis buffer, a Tween®20-based buffer, and a 1:1 acetonitrile:water mixture) for detection of heat-inactivated SARS-CoV-2 reference material (HI-SCV-2) spiked into municipal wastewater at 1.0 × 103 genomic units per millilitre (GU mL−1). The highest mean RNA concentrations were achieved using the cheesecloth (7.0 × 104 ± 3.7 × 104 GU per eluate) and electronegative filters (2.3 × 104 ± 2.5 × 104 GU per eluate) in combination with the Tween®20-based buffer with positive detections in all three biological replicates for both material types. We deployed passive samplers at two sewer catchments (locations A and B) to compare the performance of each passive sampler material type in the field. Over 15 sampling events at each site, we demonstrated that both cheesecloth (location A) and electronegative filters (location B) coupled with a Tween®20-based elution technique could be utilized for the reliable detection of SARS-CoV-2. These results have demonstrated a quick and effective passive sampling approach for SARS-CoV-2 detection in targeted locations in wastewater collection systems, which may have long-term applicability as global vaccination programs evolve.

Wastewater-based surveillance methods have been implemented in several countries as a tool for monitoring SARS-CoV-2 at a community scale. A variety of methods have been used for concentrating, extracting, and detecting the virus, with no clear consensus on the most effective approach. In this note, we report preliminary findings from a study that is tracking SARS-CoV-2 in wastewater in Halifax, Nova Scotia, with a specific focus on the use of four reverse transcriptase quantitative PCR (RT-qPCR) assays for detecting the virus in wastewater. We were able to detect the virus in wastewater samples during the initial rise of cases in the Halifax region in early November 2020. Levels of the targeted SARS-CoV-2 gene fragments increased and fell in response to reported cases of COVID-19. The CDC N1 and E RT-qPCR assays demonstrated greater relative sensitivity than the CDC N2 and N3 assays for detection of SARS-CoV-2 in raw sewage samples.

Bench-scale UV experiments are an inefficient and laborious process when using current flow-through and collimated beam technologies. Current technologies are also limited in their ability to custo...

In developing an effective monitoring program for the wastewater surveillance of SARS-CoV-2 ribonucleic acid (RNA), the importance of sampling methodology is paramount. Passive sampling has been shown to be an effective tool to detect SARS-CoV-2 RNA in wastewater. However, the adsorption characteristics of SARS-CoV-2 RNA on passive sampling material are not well-understood, which further obscures the relationship between wastewater surveillance and community infection. In this work, adsorption kinetics and equilibrium characteristics were evaluated using batch-adsorption experiments for heat-inactivated SARS-CoV-2 (HI-SCV-2) adsorption to electronegative filters. Equilibrium isotherms were assessed or a range of total suspended solids (TSS) concentrations (118, 265, and 497 mg L–1) in wastewater, and a modeled qmax of 7 × 103 GU cm–2 was found. Surrogate adsorption kinetics followed a pseudo-first-order model in wastewater with maximum concentrations achieved within 24 h. In both field and isotherm experiments, equilibrium behavior and viral recovery were found to be associated with wastewater and eluate TSS. On the basis of the results of this study, we recommend a standard deployment duration of 24–48 h and the inclusion of eluate TSS measurement to assess the likelihood of solids inhibition during analysis., Field and bench-scale studies showed that a 24−48 h deployment was ideal for a passive sampling of SARS-CoV-2 in wastewater and that analysis should include eluatimg TSS measurement to understand the role of inhibition in high TSS samples.

A commercially-available UV surface disinfection system used for hospital room disinfection was characterized for use as a UV-inactivation system for N95 filtering facepiece respirator (FFR) respirators. The output of light was initially characterized and assessed for its ability to penetrate through the individual N95 respirator layers. Following characterization, disinfection performance was tested using a range of model pathogens. In a series of experiments, coupons of respirator material were inoculated with Escherichia coli, Staphylococcus aureus, Geobacillus stearothermophilus spores, Bacillus cereus, Pseudomonas aeruginosa, and influenza A virus. Inoculated coupons were then treated with UV light from a low-pressure UV disinfection system during specific treatment times. Microbial enumeration was performed pre- and post-UV treatment. It was found that respirator coupons inoculated with E. coli, influenza A, P. aeruginosa, B. cereus and G. stearothermophilus resulted in below detection counts when exposed to UV fluences between 410 to 2700 mJ cm−2. In addition, UV light was unable to sufficiently penetrate all layers of respirator material, highlighting the importance of flipping respirators when exposing them to UV light. This work suggests that with further validation, UV light will be a promising touchless treatment option for frontline professionals who need to repurpose N95 respirators for subsequent use.

Human serum and urine samples were analyzed for a suite of nitrosatable pesticides and potentially carcinogenic pesticide-associated N-nitroso (PANN) compounds. Formation of PANN compounds may occur in vivo after consumption of food or water containing trace amounts of nitrosatable pesticide residues and nitrate. Using a modified version of the Quick, Easy, Cheap, Effective, Rugged, and Safe (QuEChERS) method, nine nitrosatable pesticides and byproducts were extracted from serum and urine from 64 individuals from two different sample populations in Atlantic Canada: (i) Prince Edward Island, a region where nitrate and trace amounts of nitrosatable pesticides have been detected in groundwater; and (ii) Halifax, Nova Scotia, a non-agricultural urban area. Samples were then analyzed using ultra-high pressure liquid chromatography (UHPLC) coupled with high-resolution accurate mass (HRAM) single-stage orbitrap mass spectrometry (MS), which allows for semi-targeted analysis and tentative identification of a virtually limitless number of exposure biomarkers. Two nitrosatable target analytes, ethylenethiourea (ETU) and 3,5,6-trichloro-2-pyridinol (TCPy) were found in serum, while atrazine (ATR) and ETU were detected in urine. Five and six PANN compounds were tentatively identified in serum and urine, respectively. The two PANN compounds that were most frequently tentatively identified in serum were N-nitroso dimethoate (N-DIM) and N-nitroso omethoate (N-OME) with detection frequencies of 78% and 95%, respectively. This is the first biomonitoring study of its kind to investigate PANN compounds in human serum and urine.

The recent surge in the use of UV technology for personal protective equipment (PPE) has created a unique learning opportunity for the UV industry to deepen surface disinfection knowledge, especially on surfaces with complex geometries, such as the N95 filter facepiece respirators (FFR). The work outlined in this study addresses the interconnectedness of independent variables (e.g., UV Fluence, respirator material) that require consideration when assessing UV light efficacy for disinfecting respirators. Through electron microscopy and Fourier-transform infrared (FTIR) spectroscopy, we characterized respirator filter layers and revealed that polymer type affects disinfection efficacy. Specifically, FFR layers made from polypropylene (PP) (hydrophobic in nature) resulted in higher disinfection efficiency than layers composed of polyethylene terephthalate (PET-P) (hygroscopic in nature). An analysis of elastic band materials on the respirators indicated that silicone rubber-based bands achieved higher disinfection efficiency than PET-P bands and have a woven, fabric-like texture. While there is a strong desire to repurpose respirators, through this work we demonstrated that the design of an appropriate UV system is essential and that only respirators meeting specific design criteria may be reasonable for repurposing via UV disinfection.

The use of magnetic beads in the extraction of nucleic acids from wastewater is presented as an approach to simplify extraction techniques for the detection of SARS-CoV-2 viral fragments in wastewater. In particular, this paper describes the development and optimization of a direct method for extracting SARS-CoV-2 RNA from municipal wastewater using magnetic beads. The recovery efficiency of the method using Accuplex SARS-CoV-2 Positive Reference Material (ASCV-2) was examined. Method factors assessed were sample volume, concentration of magnetic bead mix, elution temperature, and water matrix (deionized (DI) water and wastewater). The combination of optimized method parameters that resulted in the highest RNA recovery in both DI water (26.0 ± 0.8%) and wastewater (11.8 ± 1.4%) was a sample volume of 1.0 mL, a magnetic beads concentration of 100 µL mL-1 sample, and an elution temperature of 60 °C. The performance of this optimized method was further assessed in recovery experiments using wastewater samples spiked at 1.8×106 and 1.8×104 gene copies L-1 (GU L-1) with Gamma Inactivated SARS-COV-2 (GI-SCV-2) and 1.0×106 and 1.0×104 infectious units L-1 of Human Coronavirus 229E (HCV 229E) as viral surrogates. Recoveries of 86.1 and 4.6% were achieved for wastewater samples spiked with GI-SCV-2 at low and high concentrations, respectively. In assessing the effects of wastewater pre-filtration and addition of DL-Dithiothreitol (DTT, used to inactivate RNases that may degrade RNA) on recovery efficiency of ASCV-2, the magnetic bead-based extraction protocol performed optimally with unfiltered wastewater without DTT (recovery = 17.4 ± 0.4%). The method limit of detection (MLOD) for ASCV-2 recovered from pre-filtered wastewater was determined to be 4.6×104 GU L-1 (95% degree of confidence). Using this optimized magnetic bead-based extraction protocol, the presence of SARS-CoV-2 RNA was verified in wastewater collected from sewershed locations in Atlantic Canada. This emerging RNA extraction method is direct, rapid, and does not require the use of specialized equipment, thus offering advantageous application for laboratories with limited resources. As such, this method is an indispensable tool in the monitoring of wastewater for SARS-CoV-2 to potentially understand COVID-19 infection occurrence within communities and inform public health leaders.Graphical Abstract

N-nitroso compounds form from the interaction between nitrosatable precursors and nitrite under acidic conditions. A majority of N-nitroso compounds tested show evidence of carcinogenicity in animal models. Formation of N-nitroso compounds may occur from exposure to precursors in drinking water, but the extent of formation depends on a number of factors, including concentration of substrates, presence of catalysts and inhibitors, and pH. The objective of this study was to examine these factors in pesticide-associated N-nitroso (PANN) compound formation in drinking water. In preliminary screening experiments, nine nitrosatable pesticides and degradation products were individually reacted at environmentally-relevant concentrations (≤ 20 μg L‐−1) with sodium nitrite (NaNO2) and hydrochloric acid (HCl) in ultra-pure water. Only ethylenethiourea (ETU) showed evidence of PANN compound formation in initial experiments and was further tested for N-nitrosoethylenethiourea (N-ETU) formation in a pooled groundwater sample (comprised of five tap water samples combined into one homogenous sample) collected from an agricultural region of Prince Edward Island in Canada, where nitrate contamination is a known concern. Evidence of N-ETU formation in the groundwater sample was observed within 30 min at concentrations 7.5, 10, and 20 μg L−1. Analysis of target compounds and semi-target PANN compounds was performed using ultra-high pressure liquid chromatography coupled with high resolution orbital ion trap mass spectrometry. These preliminary experiments serve to inform about potential PANN compound formation in groundwater. The results of this study suggest that ETU is capable of forming potentially carcinogenic N-ETU in water containing nitrite/nitrate at trace concentrations under acidic conditions. Thus, these findings suggest that N-ETU formation may be a concern for individuals exposed to low concentrations of ETU in groundwater.

The use of 17β-estradiol (E2) in aquaculture feed increases growth and promotes feminization in freshwater eels. E2 and its metabolites (estrone (E1) and estriol (E3)) must be accurately measured in solid and liquid waste streams to ensure proper management. For reliable quantitation of estrogens in complex aquaculture waste solids, a rigorous clean-up method is imperative. The focus of this study was to assess, optimize, and validate a quick, easy, cheap, effective, rugged, and safe (QuEChERS) technique for extraction of estrogens from aquaculture waste solids and quantitation via liquid chromatography tandem-mass spectrometry (LC-MS/MS). Waste solids were collected from a recirculating aquaculture system containing American eels. Waste solids samples (2 g) were spiked to 50 μg kg−1 of E1, E2, and E3 and processed using 800 mg magnesium sulfate (MgSO4) and 400 mg of sodium acetate (NaOAc) as extraction salts. Two dispersive solid phase extraction (dSPE) steps were implemented to achieve optimal E2 recovery efficiency (RE (%)) in preliminary experiments (RE (%) = 102%; RSD = 5%). RE (%) for E1 and E3 were 87% (RSD = 54%) and 60% (RSD = 83%), respectively. Method performance characteristics were further assessed for E2 only and included specificity, linearity, accuracy (RE (%) and process efficiency (PE (%))), precision (intra-day precision, and inter-day precision), matrix effects, method detection limit (MDL), and limit of quantitation (LOQ). MDL and LOQ values for E2 were 0.7 and 2.3 μg kg−1, respectively. PE (%) and RE (%) were excellent (99–101% and 99–100%, respectively) and matrix effects were minimal (99 and 101%). The results of this study indicate that this method can be used for quantitation of E2 in aquaculture waste solids.

BACKGROUNDIn late 2016, US diplomats stationed in Havana began presenting with a variety of neurological manifestations that proved difficult to diagnose. Though previous studies suggested a likely association with brain injury, the mechanism of injury, brain regions involved, and etiology remained unknown.METHODSWe conducted a multimodal study examining 26 Canadian diplomats and their family members, the majority of whom presented with symptoms similar to their American counterparts while residing in Havana. Assessments included a medical history, self-reported symptom questionnaires, cognitive assessments, blood tests, and brain imaging assessments (magnetic resonance imaging (MRI) and magnetoencephalography (MEG)). Individuals showing signs of brain injury underwent further neurological, visual, and audio-vestibular assessments. Eight participants were tested both before and after living in Havana.RESULTSOur assessment documents multiple functional and structural impairments, including significant spatial memory impairment, abnormal brain-stem evoked potentials, degradation of fibre tracts in the fornix and posterior corpus callosum, blood-brain barrier injury to the right basal forebrain and anterior insula, and abnormal paroxysmal slowing events of cortical activity. Subsequent mass-spectrometry and blood analyses documented reduced serum cholinesterase activity and the presence of organophosphates (Temephos) and pyrethroid metabolites (3-phenoxybenzoic acid or 3-BPA).CONCLUSIONSOur findings confirm brain injury, specify the regions involved, and raise the hypothesis of overexposure to cholinesterase inhibitors as a plausible etiology. If correct, our hypothesis bears public health ramifications (see Discussion) and suggests a course of action for reducing exposure in the future.FUNDINGGlobal Affairs Canada.

In the acidic environment of the stomach, nitrosatable pesticide residues may react with nitrite to form potentially carcinogenic pesticide-associated N-nitroso compounds (PANOCs). The objective of this study was to develop a method for the analysis of 10 nitrosatable pesticides and breakdown products in human serum and urine. Three sample preparation methods were evaluated for extraction of target analytes from the biomatrices. Deproteinization by methanol for 300-μL aliquots of serum with a final extract volume of 225 μL resulted in excessive ion enhancement of some analytes and suppression of others. Three types of solid-phase extraction cartridges were tested for optimal analyte retention from 200-μL aliquots of serum with a final extract volume of 400 μL; this approach resulted in significant analyte loss for some compounds. The Quick, Easy, Cheap, Effective, Rugged, and Safe approach resulted in a suitable method for extraction of the analytes from each biomatrix. Biofluid samples (500 μL) were spiked to 100 μg L−1 with analytical standards and extracted using 500 μL of acetonitrile (ACN) with 4% acetic acid (AcOH) for serum and 0.1% AcOH in ACN for urine. For extraction, 200 mg magnesium sulfate (MgSO4) and 50 mg sodium acetate were added for serum and 200 mg MgSO4 and 50 mg sodium chloride were added for urine. Final extract volumes for both biomatrices using the QuEChERS method was 400 μL after dilution. Samples were analyzed via ultra-high pressure liquid chromatography/high-resolution accurate mass orbital ion trap mass spectrometry. Mean recoveries for target analytes in serum and urine ranged between 74 and 120% (%RSD

The traditional approach to extracting estrogens from water matrices, solid-phase extraction (SPE), presents a number of challenges when applied to complex wastewater matrices. Conversely, the QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe) clean-up method offers an alternative sample preparation approach that omits sample filtration and overcomes additional challenges associated with SPE. The objective of this study was to implement and validate a scaled QuEChERS method, using a standard addition approach, for extracting estrone (E1), 17β-estradiol (E2), and estriol (E3) from the estrogenic influent of a recirculating aquaculture system containing American eels (Anguilla rostrata). While traditional QuEChERS protocols do not facilitate considerable sample concentration, a 500-fold concentration factor was implemented for reliable quantitation of parts-per-trillion concentrations of estrogens from an initial sample volume of 20 mL to a final extract volume of 40 μL. Following analysis via high-performance liquid chromatography-mass spectrometry, excellent process efficiencies were observed at spiked concentrations of 10 and 50 ng L−1 for E2 and E1 (101 to 111%; %RSD ≤ 16), and moderate to acceptable process efficiencies were achieved for E3 (75 to 87%; %RSD ≤ 16). Validation of method parameters, including specificity, linearity, accuracy (recovery and process efficiencies), precision (intra-day precision, and inter-day precision), matrix effects, method detection limit, and limit of quantitation, led to reliable quantitation of unknown concentrations of E1, E2, and E3 in the aquaculture influent as low as 52, 20, and 33 ng L−1, respectively. This study provides a validated analytical method for waste systems requiring quantitation of estrogens in their complex wastewater matrices.