Background Wastewater-based epidemiology (WBE) for the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can be an important source of information for coronavirus disease 2019 (COVID-19) management during and after the pandemic. Currently, governments and transportation industries around the world are developing strategies to minimise SARS-CoV-2 transmission associated with resuming activity. This study investigated the possible use of SARS-CoV-2 RNA wastewater surveillance from airline and cruise ship sanitation systems and its potential use as a COVID-19 public health management tool. Methods Airline and cruise ship wastewater samples (n = 21) were tested for SARS-CoV-2 RNA using two virus concentration methods, adsorption-extraction by electronegative membrane (n = 13) and ultrafiltration by Amicon (n = 8), and five assays using reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and RT-droplet digital PCR (RT-ddPCR). Representative amplicons from positive samples were sequenced to confirm assay specificity. Results SARS-CoV-2 RNA was detected in samples from both aircraft and cruise ship wastewater; however, concentrations were near the assay limit of detection. The analysis of multiple replicate samples and use of multiple RT-qPCR and/or RT-ddPCR assays increased detection sensitivity and minimised false-negative results. Representative amplicons were confirmed for the correct PCR product by sequencing. However, differences in sensitivity were observed among assays and concentration methods. Conclusions The study indicates that surveillance of wastewater from large transport vessels with their own sanitation systems has potential as a complementary data source to prioritize clinical testing and contact tracing among disembarking passengers. Importantly, sampling methods and molecular assays must be further optimized to maximize sensitivity. The potential for false negatives by both wastewater testing and clinical swab testing suggests that the two strategies could be employed together to maximize the probability of detecting SARS-CoV-2 infections amongst passengers.

The in-sample stability of selected pharmaceuticals, illicit drugs, and their metabolites in wastewater was assessed under six different conditionsuntreated, addition of hydrochloric acid or sodium metabisulfite solution, combined with or without sterile filtration, and at four representative temperatures, at 35 °C for up to 28 days, 22 °C for 56 days, and 4 °C and −20 °C for 196 days, or freeze/thaw cycles for 24 weeks. Paracetamol, 6-monoacetylmorphine, morphine, and cocaine were poorly stable in untreated wastewatere.g., with 50% transformation within 1.2−8.1 days at 22 °C, and acidification reduced their in-sample transformations. Acesulfame, carbamazepine, cotinine, methamphetamine, 3,4methylenedioxy-methamphetamine (MDMA), ketamine, norfentanyl, 3,4-methylenedioxy-N-ethylamphetamine (MDEA), and norbuprenorphine were highly or moderately stable over the observed period, even in untreated wastewater. Fitting of pseudofirst-order kinetics and the Arrhenius equation was used to develop a multistage transformation estimation model combined with an interactive tool to evaluate possible transformation scenarios of selected biomarkers for the processes from sampling to preanalysis. However, as the wastewater composition can vary between sites and over time, the variability of in-sample stability requires further exploration.

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Wastewater-based epidemiology is a potential complementary technique for monitoring the use of performanceand image-enhancing drugs (PIEDs), such as anabolic steroids and selective androgen receptor modulators (SARMs), within the general population. Assessing in-sewer transformation and degradation is critical for understanding uncertainties associated with wastewater analysis. An electrospray ionization liquid chromatography mass spectrometry method for the quantification of 59 anabolic agents in wastewater influent was developed. Limits of detection and limits of quantification ranged from 0.004 to 1.56 μg/L and 0.01 to 4.75 μg/L, respectively. Method performance was acceptable for linearity (R 2 > 0.995, few exceptions), accuracy (68−119%), and precision (1−21%RSD), and applicability was successfully demonstrated. To assess the stability of the selected biomarkers in wastewater, we used laboratory-scale sewer reactors to subject the anabolic agents to simulated realistic sewer environments for 12 h. Anabolic agents, including parent compounds and metabolites, were spiked into freshly collected wastewater that was then fed into three sewer reactor types: control sewer (no biofilm), gravity sewer (aerobic conditions), and rising main sewer (anaerobic conditions). Our results revealed that while most glucuronide conjugates were completely transformed following 12 h in the sewer reactors, 50% of the investigated biomarkers had half-lives longer than 4 h (mean residence time) under gravity sewer conditions. Most anabolic agents were likely subject to biofilm sorption and desorption. These novel results lay the groundwork for any future wastewater-based epidemiology research involving anabolic steroids and SARMs.