Influent Wastewater Samples Research Articles

Drug Use Patterns in Wastewater and Socioeconomic and Demographic Indicators

Measuring drug use behaviors in individuals and across large communities presents substantial challenges, often complicated by socioeconomic and demographic variables. To detect spatial and temporal changes in community drug use by analyzing concentrations of analytes in influent wastewater and exploring their associations with area-based socioeconomic and sociodemographic metrics like the area deprivation index (ADI) and rural-urban commuting area (RUCA) codes. This longitudinal, cross-sectional wastewater study was performed from May 2022 to April 2023 and included biweekly influent wastewater samples of 39 analytes from 8 sampling locations across 6 wastewater treatment plants in southern Nevada. Statistical analyses were conducted in December 2023. It was hypothesized that wastewater monitoring of pharmaceuticals and personal care products (PPCPs) and high-risk substances (HRSs) could reveal true spatial and temporal drug use patterns in near-real time. Data collection of samples for PPCPs and HRSs was performed using mass spectrometry. Both ADI and RUCA scores were utilized to characterize neighborhood contexts in the analysis. The false discovery rate (FDR) method was utilized to correct for multiple comparisons (PFDR). Over the 12-month wastewater monitoring period, 208 samples for PPCPs and HRSs were collected, and analysis revealed an increase in the consumption of HRSs and the seasonal variation in PPCP use in southern Nevada. There was a significant increase in levels of stimulant-associated analytes, such as cocaine (β = 9.17 × 10-4; SE = 1.29 × 10-4; PFDR = 1.40 × 10-10), and opioids or their metabolites, notably norfentanyl (β = 1.48 × 10-4; SE = 1.88 × 10-4; PFDR = 1.66 × 10-12). In contrast, DEET, an active ingredient in mosquito and tick repellents, demonstrated a seasonal use pattern (β = -4.85 × 10-4; SE = 2.09 × 10-4; PFDR = 4.87 × 10-2). Wastewater from more disadvantaged or rural neighborhoods, as assessed through ADI and RUCA scores, was more likely to show a significant positive correlation with HRSs, such as cocaine (β = 0.075; SE = 0.038; P = .05) and norfentanyl (β = 0.004; SE = 0.001; P = 1.64 × 10-5). These findings suggest that wastewater monitoring of PPCPs and HRSs offers a complementary method to existing public health tools, providing timely data for tracking substance use behaviors and use of PPCPs at a population level.

A key factor in monitoring cannabis consumption trends through wastewater analysis: Partitioning of THC[sbnd]COOH between the liquid and solid phase of influent wastewater

Current wastewater-based epidemiology (WBE) studies are predominantly focused on the analysis of urinary biomarkers present in the liquid phase of influent wastewater (IWW). This approach systematically underestimates less polar metabolites, such as cannabis biomarkers. These biomarkers can potentially sorb to and desorb from suspended particulate matter (SPM) present in IWW. This study investigates the bidirectional partitioning of THCCOOH between the liquid phase and SPM of IWW by performing multiple sorption experiments using THCCOOH-D9 as a surrogate due to the unavailability of blank SPM and blank IWW. In addition, this study involves the analysis of IWW collected from eight wastewater treatment plants (WWTP) (n = 56) across four European countries, where raw IWW, the liquid phase and SPM were separately analysed to identify potential trends in the fraction of THCCOOH in the SPM between and withing the examined locations. Based on the performed sorption experiments, bidirectional partitioning of THCCOOH between the liquid phase and SPM was noted, showing partition between both phases when only one of the two phases was spiked. It was illustrated that the concentration of SPM had a notable influence on the THCCOOH partitioning between both phases. In addition to the inter location variability (average THCCOOH present in the SPM ranged 31–59 %), a substantial intra location variation was also observed, e.g., in one location ranged 17–58 %. While the determination of a correction factor for the amount of THCCOOH present in SPM would be ideal, this is challenging, since the amount of SPM is not fixed in all IWW samples. Although SPM has influence on the THCCOOH partition, no correlation (p value Spearman correlation = 0.3160) was observed between the SPM concentration and the fraction of THCCOOH in the solid phase. Moreover, the collection of homogenized samples is difficult, and the time required to reach an equilibration in partitioning of THCCOOH between both IWW phases remains unclear. Due to i) the large inter-and intra-location variation of THCCOOH present in the SPM, ii) the variability in SPM concentration in IWW samples, and iii) the time required to reach a partitioning equilibration, an analytical procedure based on liquid-liquid extraction (LLE) that considers both the liquid phase and SPM of IWW is recommended to reduce the overall uncertainty for THCCOOH measurement in IWW. It was illustrated that this extraction method is capable to recover the total concentration of THCCOOH in both phases.

Wastewater-based epidemiology to assess pharmaceutical consumption. Spanish perspective

Wastewater-based epidemiology (WBE) is a well-established approach that can provide objective and real-time data on the consumption of substances such as pharmaceuticals. However, most of the studies reported so far compares consumption data obtained using WBE with those derived from prescription data from public health systems, which is often incomplete and might represent a source of uncertainty.This study aims to compare the measured pharmaceutical consumption back calculated with the WBE approach with consumption derived from dispensed pharmaceuticals in two regions of Spain, managed by two different Health Systems. To do so, a group of 17 pharmaceuticals, including the most representative ones of every therapeutic family, were monitored in influent wastewater (IWW) samples collected over a week campaign in spring 2022 at four different wastewater treatment plants (WWTPs) in Spain: two WWTPs in Madrid city (center of Spain) and two WWTPs in Catalonia (Northeast of Spain).Population-normalized daily loads (PNDL) revealed that the patterns of pharmaceutical occurrence in the different WWTPs are very similar, being acetaminophen, 4-acetamidoantipyrine and valsartan the pharmaceuticals with the highest PNDL values: 17162 ± 1457 mg day−1 1000 inh−1 for acetaminophen, 2365 ± 696 and 2429 ± 263 mg day−1 1000 inh−1 for 4-acetamidoantipyrine, 2006 ± 541 and 2041 ± 352 mg day−1 1000 inh−1 for valsartan.Pharmaceutical PNLDs were then transformed into measured pharmaceutical consumption (MC) and compared with dispensed consumption (DC) data obtained from the pharmacies in the catchment area where the WWTPs are located. A ratio MC/DC within 0.8 to 1.2 was obtained for 11 out of the 17 studied pharmaceuticals. Highlighting a match in all the cardiovascular system pharmaceuticals, with the exception of losartan (1.29–1.39 ratio) and valsartan (1.35–1.43) in all WWTPs. In summary, the degree of correlation between MC/DC is higher than those previously reported comparing with the prescribed pharmaceutical consumption.

Wastewater-based surveillance of SARS-CoV-2: Short-term projection (forecasting), smoothing and outlier identification using Bayesian smoothing

BackgroundDay-to-day variation in the measurement of SARS-CoV-2 in wastewater can challenge public health interpretation. We assessed a Bayesian smoothing and forecasting method previously used for surveillance and short-term projection of COVID-19 cases, hospitalizations, and deaths. MethodsSARS-CoV-2 viral measurement from the sewershed in Ottawa, Canada, sampled at the municipal wastewater treatment plant from July 1, 2020, to February 15, 2022, was used to assess and internally validate measurement averaging and prediction. External validation was performed using viral measurement data from influent wastewater samples from 15 wastewater treatment plants and municipalities across Ontario. ResultsPlots of SARS-CoV-2 viral measurement over time using Bayesian smoothing visually represented distinct COVID-19 “waves” described by case and hospitalization data in both initial (Ottawa) and external validation in 15 Ontario communities. The time-varying growth rate of viral measurement in wastewater samples approximated the growth rate observed for cases and hospitalization. One-week predicted viral measurement approximated the observed viral measurement throughout the assessment period from December 23, 2020, to August 8, 2022. An uncalibrated model showed underprediction during rapid increases in viral measurement (positive growth) and overprediction during rapid decreases. After recalibration, the model showed a close approximation between observed and predicted estimates. ConclusionBayesian smoothing of wastewater surveillance data of SARS-CoV-2 allows for accurate estimates of COVID-19 growth rates and one- and two-week forecasting of SARS-CoV-2 in wastewater for 16 municipalities in Ontario, Canada. Further assessment is warranted in other communities representing different sewersheds and environmental conditions.

A novel Z-scheme covalent triazine framework/silver phosphate (CTF/Ag3PO4) heterojunction photocatalyst: Improved adsorption, photocatalytic degradation and degradation mechanism of rifampicin

In order to achieve a suitable covalent triazine framework (CTF) photocatalyst, it is imperative to further improve synthesis conditions of ionothermal trimerization technique. Therefore, in this study, CTF was prepared using a binary ZnCl2-NaCl molten salt mixture at 250 °C for 20 h. Furthermore, a Z-scheme CTF/silver phosphate (CTF/Ag3PO4) was prepared by facile in-situ precipitation technique for the adsorption and photocatalytic degradation of rifampicin (RIF) under visible light. The synthesized CTF, Ag3PO4, and CTF/Ag3PO4 composites were extensively characterized using various spectroscopic tools. The composites displayed improved visible light absorption, low resistance to charge transfer, and enhanced heteroatom effect (HAE) compared to pristine CTF and Ag3PO4. The CTF/Ag3PO4 composite showed improved rifampicin removal from solution. Consequently, the 0.75 % CTF/Ag3PO4 composite attained an adsorption capacity of 41.72 mg/g and 100 % degradation of RIF, which corresponded to a 3.5-fold improvement compared to pristine CTF. Its efficiency was attributed to its narrow band gap of 2.20 eV, improved surface area, and low resistance to charge transfer. Degradation product analysis revealed that rifampicin with m/z of 823 was successfully degraded to m/z of 189 within 360 min. The successful removal of RIF from influent wastewater (65.5 %) and tap water (75.5 %) samples indicates the practical application of 0.75 % CTF/Ag3PO4. The stability measurement suggests a stable 0.75 % CTF/Ag3PO4 material compared to Ag3PO4.

Are we using more sugar substitutes? Wastewater analysis reveals differences and rising trends in artificial sweetener usage in Swedish urban catchments

The market for artificial sweeteners as substitutes for conventional sugar (sucrose) is growing, despite potential health risks associated with their intake. Estimating population usage of artificial sweeteners is therefore crucial, and wastewater analysis can serve as a complement to existing methods. This study evaluated spatial and temporal usage of artificial sweeteners in five Swedish communities based on wastewater analysis. We further compared their levels measured in wastewater with the restrictions during the COVID-19 pandemic in Sweden and assessed health risks to the Swedish population. Influent wastewater samples (n = 194) collected in March 2019-February 2022 from communities in central and southern Sweden were analyzed for acesulfame, saccharin, and sucralose using liquid-chromatography coupled with tandem mass spectrometry. Spatial differences in loads for individual artificial sweetener were observed, with sucralose being higher in Kalmar (southern Sweden), and acesulfame and saccharin in Enköping and Östhammar (central Sweden). Based on sucrose equivalent doses, all communities showed a consistent prevalence pattern of sucralose > acesulfame > saccharin. Four communities with relatively short monitoring periods showed no apparent temporal changes in usage, but the four-year monitoring in Uppsala revealed a significant (p < 0.05) annual increase of ∼19 % for sucralose, ∼9 % for acesulfame and ∼8 % for saccharin. This trend showed no instant or delayed effects from COVID-19 restrictions, reflecting positively on the studied population which retained similar exposure to the artificial sweeteners despite potential pandemic stresses. Among the three artificial sweeteners, only acesulfame’s levels were at the lower end of the health-related threshold for consumption of artificially sweetened beverages; yet, all were far below the acceptable daily intake, indicating no appreciable health risks. Our study provided valuable, pilot insights into the spatio-temporal usage of artificial sweeteners in Sweden and their associated health risks. This shows the usefulness of wastewater analysis for public health authorities wishing to assess future relevant interventions.

Application of Skimmed-Milk Flocculation Method for Wastewater Surveillance of COVID-19 in Kathmandu, Nepal.

Wastewater surveillance (WS) has been used globally as a complementary tool to monitor the spread of coronavirus disease 2019 (COVID-19) throughout the pandemic. However, a concern about the appropriateness of WS in low- and middle-income countries (LMICs) exists due to low sewer coverage and expensive viral concentration methods. In this study, influent wastewater samples (n = 63) collected from two wastewater treatment plants (WWTPs) of the Kathmandu Valley between March 2021 and February 2022 were concentrated using the economical skimmed-milk flocculation method (SMFM). The presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was tested by qPCR using assays that target the nucleocapsid (N) and envelope (E) genes. Overall, 84% (53/63) of the total samples were positive for SARS-CoV-2 according to at least one of the tested assays, with concentrations ranging from 3.5 to 8.3 log10 gene copies/L, indicating the effectiveness of the SMFM. No correlation was observed between the total number of COVID-19 cases and SARS-CoV-2 RNA concentrations in wastewater collected from the two WWTPs (p > 0.05). This finding cautions the prediction of future COVID-19 waves and the estimation of the number of COVID-19 cases based on wastewater concentration in settings with low sewer coverage by WWTPs. Future studies on WS in LMICs are recommended to be conducted by downscaling to sewer drainage, targeting a limited number of houses. Overall, this study supports the notion that SMFM can be an excellent economical virus-concentrating method for WS of COVID-19 in LMICs.

Wastewater-based surveillance is an efficient monitoring tool for tracking influenza A in the community

Around the world, influenza A virus has caused severe pandemics, and the risk of future pandemics remains high. Currently, influenza A virus surveillance is based on the clinical diagnosis and reporting of disease cases. In this study, we apply wastewater-based surveillance to monitor the amount of the influenza A virus RNA at the population level. We report the influenza A virus RNA levels in 10 wastewater treatment plant catchment areas covering 40 % of the Finnish population. Altogether, 251 monthly composite influent wastewater samples (collected between February 2021 and February 2023) were analysed from supernatant fraction using influenza A virus specific RT-qPCR method. During the study period, an influenza A virus epidemic occurred in three waves in Finland. This study shows that the influenza A virus RNA can be detected from the supernatant fraction of 24 h composite influent wastewater samples. The influenza A virus RNA gene copy number in wastewater correlated with the number of confirmed disease cases in the Finnish National Infectious Diseases Register. The median Kendall's τ correlation strength was 0.636 (min= 0.486 and max=0.804) and it was statistically significant in all 10 WTTPs. Wastewater-based surveillance of the influenza A virus RNA is an independent from individual testing method and cost-efficiently reflects the circulation of the virus in the entire population. Thus, wastewater monitoring complements the available, but often too sparse, information from individual testing and improves health care and public health preparedness for influenza A virus pandemics.

Urban wastewater-based epidemiology for multi-viral pathogen surveillance in the Valencian region, Spain

Wastewater-based epidemiology (WBE) has lately arised as a promising tool for monitoring and tracking viral pathogens in communities. In this study, we analysed WBE's role as a multi-pathogen surveillance strategy to detect the presence of several viral illness causative agents. Thus, an epidemiological study was conducted from October 2021 to February 2023 to estimate the weekly levels of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Respiratory Syncytial virus (RSV), and Influenza A virus (IAV) in influent wastewater samples (n = 69). In parallel, a one-year study (October 2021 to October 2022) was performed to assess the presence of pathogenic human enteric viruses. Besides, monitoring of proposed viral fecal contamination indicators crAssphage and Pepper mild mottle virus (PMMoV) was also assessed, along with plaque counting of somatic coliphages. Genetic material of rotavirus (RV), human astrovirus (HAStV), and norovirus genogroup I (GI) and GII was found in almost all samples, while hepatitis A and E viruses (HAV and HEV) only tested positive in 3.77 % and 22.64 % of the samples, respectively. No seasonal patterns were overall found for enteric viruses, although RVs had a peak prevalence in the winter months. All samples tested positive for SARS-CoV-2 RNA, with a mean concentration of 5.43 log genome copies per liter (log GC/L). The tracking of the circulating SARS-CoV-2 variants of concern (VOCs) was performed by both duplex RT-qPCR and next generation sequencing (NGS). Both techniques reliably showed how the dominant VOC transitioned from Delta to Omicron during two weeks in Spain in December 2021. RSV and IAV viruses peaked in winter months with mean concentrations 6.40 and 4.10 log GC/L, respectively. Moreover, the three selected respiratory viruses strongly correlated with reported clinical data when normalised by wastewater physico-chemical parameters and presented weaker correlations when normalising sewage concentration levels with crAssphage or somatic coliphages titers. Finally, predictive models were generated for each respiratory virus, confirming high reliability on WBE data as an early-warning system and communities illness monitoring system. Overall, this study presents WBE as an optimal tool for multi-pathogen tracking reflecting viral circulation and diseases trends within a selected area, its value as a multi-pathogen early-warning tool stands out due to its public health interest.

Workflow to facilitate the detection of new psychoactive substances and drugs of abuse in influent urban wastewater

The complexity around the dynamic markets for new psychoactive substances (NPS) forces researchers to develop and apply innovative analytical strategies to detect and identify them in influent urban wastewater. In this work a comprehensive suspect screening workflow following liquid chromatography – high resolution mass spectrometry analysis was established utilising the open-source InSpectra data processing platform and the HighResNPS library. In total, 278 urban influent wastewater samples from 47 sites in 16 countries were collected to investigate the presence of NPS and other drugs of abuse. A total of 50 compounds were detected in samples from at least one site. Most compounds found were prescription drugs such as gabapentin (detection frequency 79%), codeine (40%) and pregabalin (15%). However, cocaine was the most found illicit drug (83%), in all countries where samples were collected apart from the Republic of Korea and China. Eight NPS were also identified with this protocol: 3-methylmethcathinone 11%), eutylone (6%), etizolam (2%), 3-chloromethcathinone (4%), mitragynine (6%), phenibut (2%), 25I-NBOH (2%) and trimethoxyamphetamine (2%). The latter three have not previously been reported in municipal wastewater samples. The workflow employed allowed the prioritisation of features to be further investigated, reducing processing time and gaining in confidence in their identification.

Influent wastewater analysis to investigate emerging trends of new psychoactive substances use in Europe

Wastewater-based epidemiology (WBE) can provide objective and timely information on the use of new psychoactive substances (NPS), originally designed as legal alternatives of internationally controlled drugs. NPS have rapidly emerged on the global drug market, posing a challenge to drug policy and constituting a risk to public health. In this study, a WBE approach was applied to monitor the use of more than 300 NPS, together with fentanyl and its main metabolite norfentanyl, in influent wastewater collected from 12 European cities during March-June 2021. Quantitative and qualitative analysis of NPS in composite 24 h influent wastewater samples were based on solid phase extraction and liquid chromatography-mass spectrometry. In-sample stability tests demonstrated the suitability of most investigated biomarkers, except for a few synthetic opioids, synthetic cannabinoids and phenetylamines. Fentanyl, norfentanyl and eight NPS were quantified in influent wastewater and at least three substances were found in each city, demonstrating their use in Europe. N,N-dimethyltryptamine and 3-methylmethcathinone (3-MMC) were the most common NPS found, with the latter having the highest mass loads (up to 24.8 mg/day/1000 inhabitants). Seven additional substances, belonging to five categories of NPS, were identified in different cities. Spatial trends of NPS use were observed between cities and countries, and a changing weekly profile of use was observed for 3-MMC. WBE is a useful tool to rapidly evaluate emerging trends of NPS use, complementing common indicators (i.e. population surveys, seizures) and helping to establish measures for public health protection.

Absence of new psychoactive substances in wastewater from South Wales, UK, revealed by optimised liquid chromatography-time-of-flight analysis.

New psychoactive substances are produced and marketed to mimic the effects of their illicit counterparts and to attempt to evade drug tests and prosecution. Here, we present the optimisation, validation and application of an analytical method using liquid chromatography-time-of-flight mass spectrometry to detect and quantify 37 new psychoactive substances and illicit substances in wastewater from South Wales, UK, using a targeted analysis method. Sample preparation was performed using solid-phase extraction with Oasis HLB cartridges. The LC separation was performed using a YMC-Triart Phenyl 450 bar column (12 nm, 5μm, 100 × 3mm) which provided good separation and resolution for all targeted analytes with a run time of 9min. The method was validated using the following parameters: sensitivity, selectivity, linearity, accuracy, precision, recovery and matrix effects. The method was then applied to influent wastewater samples collected from two wastewater treatment plants in Wales, UK.

Wastewater-based monitoring of the nitazene analogues: First detection of protonitazene in wastewater

Synthetic opioids, particularly the nitazene analogues class, have become a public health concern due to their high potency. Wastewater-based epidemiology can detect community use of these compounds. The objective of this work was to detect nitazene analogues in wastewater from samples collected from eight sites in the United States. Influent wastewater samples were collected from eight sites in seven states (Arizona, Oregon, New Mexico, Illinois, New Jersey, Washington and Georgia) in the United States. Samples were collected from each site on three days between 27 December 2022 and 4 January 2023, acidified on collection, stored frozen and shipped to Arizona State University (Tempe, AZ) for sample processing. Samples were then shipped to The University of Queensland (Brisbane, Australia) for sample analysis. Protonitazene was found in samples collected from two sites in Washington and Illinois. The concentration was estimated up to 0.5 ng/L, with estimated excreted mass loads up to 0.3 mg/day/1000 people. This work has shown that it is possible to detect nitazene analogues in wastewater using a combination of sample pre-concentration and sensitive instrumentation, thereby further expanding the utility of wastewater-based epidemiology.

Target and non-target screening of biomarkers in wastewater: towards a unique analytical methodology for sample preparation.

This study aims to optimize a single preparation methodology based on solid-phase extraction (SPE) that could fit both target and non-target screening of organic biomarkers in raw wastewater, allowing the cross-comparison of results obtained from a same dataset. The efficiency of SPE sorbents used alone (HLB) or in combination in a multilayer cartridge was evaluated based on (i) the extraction recovery and matrix effect in environmental samples (surface water and wastewater) for a list of biomarkers (pharmaceuticals, licit and illicit drugs, artificial sweeteners, isoprostanes, polyphenols) and (ii) a number of detected features and their intensity in HRMS. The selected method uses a combination of three SPE sorbents mixed together (HLB, X-AW and X-CW) and seems to take full advantage of each, providing satisfactory validation parameters (recovery, instrumental limit of detection, linearity range and limit of quantification) over a large range of physico-chemical properties while ensuring promising results for non-target screening applications. Of the 65 targeted compounds, nearly all of them (47) were detected in wastewater influent samples with concentration above the limit of quantification, while at the same time over 10 000 features were recorded according to the high resolution mass spectrometry (HRMS) fingerprint, holding out the promise that a common protocol for these two analyses, with their very contrasting constraints and objectives, is possible.

Adsorption Isotherms and Kinetics of Acebutolol and Metoprolol on Magnetic Nanocomposite Fe3O4@MIL‐101(Cr)

AbstractContamination of water bodies by emerging pollutants such as β‐blockers has been a global concern over the past few years. This is due to the bioaccumulative character of β‐blockers in the aquatic systems, and their excessive usage might cause adverse effects on both humans and aquatic biota. A reusable and recyclable magnetic chromium‐based MIL‐101 (Fe3O4@MIL‐101(Cr)) nanocomposite was used as an adsorbent for the removal of the selected β‐blockers (acebutolol and metoprolol) from wastewater. The nanocomposite‘s structural, magnetic and surface properties were confirmed using various characterisation techniques. The equilibrium data indicated that the maximum adsorption capacities of Fe3O4@MIL‐101(Cr) for acebutolol and metoprolol were 30.9 mg g−1 and 28.3 mg g−1, respectively. The isotherm and kinetic experimental data best fitted Langmuir isotherm and pseudo‐second‐order kinetics models. Obtained free energies using the Dubinin‐Radushkevich model were above 8 kJ mol−1, demonstrating that the interaction mechanism between β‐blockers and the Fe3O4@MIL‐101(Cr) nanocomposite was chemisorption. The prepared nanocomposite was then used to remove β‐blockers from real samples, and maximum removal efficiencies (94.1–97.1 %) were obtained in the presence of co‐existing species. The Fe3O4@MIL‐101(Cr) hybrid showed great potential for efficient and facile removal of β‐blockers from river water, effluent and influent wastewater samples.

Genomic analysis of Citrobacter from Australian wastewater and silver gulls reveals novel sequence types carrying critically important antibiotic resistance genes

Antimicrobial resistance (AMR) is a major public health concern, and environmental bacteria have been recognized as important reservoirs of antimicrobial resistance genes (ARGs). Citrobacter, a common environmental bacterium and opportunistic pathogen in humans and other animals, has been largely understudied in terms of its diversity and AMR potential. Whole-genome (short-read) sequencing on a total of 77 Citrobacter isolates obtained from Australian silver gull (Chroicocephalus novaehollandiae) (n = 17) and influent wastewater samples (n = 60) was performed, revealing a diverse Citrobacter population, with seven different species and 33 sequence types, 17 of which were novel. From silver gull using non-selective media we isolated a broader range of species with little to no mobilised ARG carriage. Wastewater isolates (selected using Carbapenem- Resistant Enterobacterales (CRE) selective media) carried a heavy burden of ARGs (up to 21 ARGs, conferring resistance to nine classes of antibiotics), with several novel multidrug-resistant (MDR) lineages identified, including C. braakii ST1110, which carried ARGs conferring resistance to eight to nine classes of antibiotics, and C. freundii ST1105, which carried two carbapenemase genes, blaIMP-4 in class 1 integron structure, and blaKPC-2. Additionally, we identified an MDR C. portucalensis isolate carrying blaNDM-1, blaSHV-12, and mcr-9. We identified IncC, IncM2, and IncP6 plasmids as the likely vectors for many of the critically important mobilised ARGs. Phylogenetic analyses were performed to assess any epidemiological linkages between isolation sources, demonstrating low relatedness across sources beyond the ST level. However, these analyses did reveal some closer relationships between strains from disparate wastewater sources despite their collection some 13,000 km apart. These findings support the need for future surveillance of Citrobacter populations in wastewater and wildlife populations to monitor for potential opportunistic human pathogens.

Selective determination of 2-aminobenzothiazole in environmental water and organic extracts from fish and dust samples

In the present study, a homemade mixed-mode ion-exchange sorbent based on silica with embedded graphene microparticles is applied for the selective extraction of 2-aminobenzothiazole (NH2BT) followed by determination through liquid chromatography coupled to high-resolution mass spectrometry. The sorbent was evaluated for the solid-phase extraction of NH2BT from environmental water samples (river, effluent wastewater, and influent wastewater), and NH2BT was strongly retained through the selective cation-exchange interactions. Therefore, the inclusion of a clean-up step of 7 mL of methanol provided good selectivity for the extraction of NH2BT. The apparent recoveries obtained for environmental water samples ranged from 62 to 69% and the matrix effect from −1 to −14%. The sorbent was also evaluated in the clean-up step of the organic extract for the extraction of NH2BT from organic extracts of indoor dust samples (10 mL of ethyl acetate from pressurized liquid extraction) and fish (10 mL of acetonitrile from QuEChERS extraction). The organic extracts were acidified (adding a 0.1% of formic acid) to promote the cation-exchange interactions between the sorbent and the analyte. The apparent recoveries for fish samples ranged from 22 to 36% depending on the species. In the case of indoor dust samples, the recovery was 41%. It should be highlighted the low matrix effect encountered in such complex samples, with values ranging from −7 to 5% for fish and dust samples. Finally, various samples were analyzed. The concentration in river samples ranged from 31 to 136 ng/L; in effluent wastewater samples, from 55 to 191 ng/L; in influent wastewater samples, from 131 to 549 ng/L; in fish samples, from 14 to 57 ng/g dried weight; and in indoor dust samples, from <MQL to 114 ng/g.

Yeast estrogen screen assay applied in the assessment of estrogenic activity removal from dairy cattle wastewater treated by anaerobic digestion

The presence of estrogen-like endocrine disrupting chemicals (EEDC) in surface waters, soils, and groundwater has been frequently reported in the literature. Since these compounds can be excreted by humans and animals, the release of domestic sewage and animal wastewaters may be an important source of EEDC in the environment. In this context, there is a growing concern regarding the presence of these substances in the environment and their potential adverse effects on human health. However, most studies have been focused on the presence of EEDC in domestic wastewaters and the available information regarding EEDC in livestock wastewater is still limited. Therefore, this work aimed to quantify the estrogenic activity (EA) in the liquid phase of dairy cattle wastewaters and evaluate the removal of EA during anaerobic digestion, applying an adapted methodology based on the yeast estrogen screen (YES) assay, which has been widely used to analyze EA in domestic sewage. Influent and effluent dairy cattle wastewater samples from a full-scale anaerobic digester placed in an experimental farm were collected during seven months. EA concentrations in the liquid phase of raw wastewater ranged from 228 to 2182 ng.L−1 E2-eq. Anaerobic digestion was not efficient in EA removal, achieving effluent concentrations ranging from 109 to 946 ng.L−1 E2-eq. Results also indicate that the application of an estradiol-based fixed time artificial insemination protocol did not statistically contribute to the increase of EA concentrations in dairy cattle wastewater which reuse in cleaning of animal feeding operation facilities and fertigation are beneficial and economically advantageous to dairy production systems, reducing water demand in parallel to promoting organic matter and nutrient recycling. However, the potential environmental and health risks associated with the presence of EEDC in dairy cattle wastewaters should be better elucidated and evaluated.

Comprehensive study on the potential environmental risk of temporal antibiotic usage through wastewater discharges

Antibiotic residues can reach aquatic ecosystems through urban wastewater discharges, posing an ecotoxicological risk for aquatic organisms and favoring the development of bacterial resistance. To assess the emission rate and hazardousness of these compounds, it is important to carry out periodic chemical monitoring campaigns that provide information regarding the actual performance of wastewater treatment plants (WWTPs) and the potential impact of the treated wastewater in the aquatic environment. In this study, 18 of the most widely consumed antibiotics in Spain were determined by liquid chromatography-tandem mass spectrometry in both influent (IWW) and effluent wastewater (EWW) samples collected over four seasons along 2021–2022. Eleven antibiotics were detected in EWW with azithromycin, ciprofloxacin and levofloxacin showing the highest concentration levels (around 2 μg L−1 of azithromycin and 0.4 μg L−1 of quinolone compounds). Data showed that only 4 out of the 11 compounds were removed by more than 50 % in the WWTP, with sulfamethoxazole standing out with an average removal efficiency >80 %. The risk that treated water could pose to the aquatic environment was also assessed, with 6 compounds indicating a potential environmental risk by exceeding established ecotoxicological and resistance thresholds. Based on the risk assessment, the WWTP removal efficiency required to reduce such risk for antibiotics was estimated. In addition, pooled wastewater samples were screened by LC coupled to high resolution mass spectrometry with ion mobility separation, searching for metabolites and transformation products of the antibiotics investigated to widen future research. Studies like this are crucial to map the impact of antibiotic pollution and to provide the basis for designing water quality and risk prevention monitoring programs.

Response of wastewater-based epidemiology predictor for the second wave of COVID-19 in Ahmedabad, India: A long-term data Perspective

In this work, we present an eight-month longitudinal study of wastewater-based epidemiology (WBE) in Ahmedabad, India, where wastewater surveillance was introduced in September 2020 after the successful containment of the first wave of COVID-19 to predict the resurge of the infection during the second wave of the pandemic. The study aims to elucidate the weekly resolution of the SARS-CoV-2 RNA data for eight months in wastewater samples to predict the COVID-19 situation and identify hotspots in Ahmedabad. A total of 287 samples were analyzed for SARS-CoV-2 RNA using RT-PCR, and Spearman's rank correlation was applied to depict the early warning potential of WBE. During September 2020 to April 2021, the increasing number of positive wastewater influent samples correlated with the growing number of confirmed clinical cases. It also showed clear evidence of early detection of the second wave of COVID-19 in Ahmedabad (March 2021). 258 out of a total 287 samples were detected positive with at least two out of three SARS-CoV-2 genes (N, ORF- 1 ab, and S). Monthly variation represented a significant decline in all three gene copies in October compared to September 2020, followed by an abrupt increase in November 2020. A similar increment in the gene copies was observed in March and April 2021, which would be an indicator of the second wave of COVID-19. A lead time of 1–2 weeks was observed in the change of gene concentrations compared with clinically confirmed cases. Measured wastewater ORF- 1 ab gene copies ranged from 6.1 x 102 (October 2020) to 1.4 x 104 (November 2020) copies/mL, and wastewater gene levels typically lead to confirmed cases by one to two weeks. The study highlights the value of WBE as a monitoring tool to predict waves within a pandemic, identify local disease hotspots within a city, and guide rapid management interventions.