Wastewater Treatment Plant Samples Research Articles

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.

Microplastics pollution from wastewater treatment plants: A critical review on challenges, detection, sustainable removal techniques and circular economy

The wastewater treatment plants (WWTPs) have long been recognized as a potentially significant source of microplastic (MPs) pollution in aquatic environments. It is challenging to precisely detect and efficiently remove MPs from wastewater due to the lack of standard protocol and removal technologies. Although many advances have been made in the characterization and quantification of MPs in wastewater, the polydisperse and degradation-resistant properties of MPs and WWTPs’ broad nature and lack of specific design allow MPs to pass through treatment processes. This review highlights the current understanding on the occurrence, fate and expulsion of MPs, recent detection status, advanced and sustainable removal technologies for MPs in WWTPs and conversion of MPs into renewable energy sources. Specifically, different techniques to collect MPs from sewage sludge and wastewater, pre-treatment, classification approaches and retention of MPs in wastewater are reviewed and analyzed. Previous MPs studies have undoubtedly improved our understanding, but many unanswered questions need to be answered. The main challenges appear to be the harmonization of detection methods and the removal levels of MPs in wastewater and sewage sludge. To address this challenge, an alternative methodological approach for WWTPs effluent sampling was developed that eliminate sample contamination by synthetic fibre deposition during aquatic sample processing. In parallel, the sustainable methodology was developed for the treatment of sewage sludge samples that effectively separates the MPs from the organic and organometal compositions of the sludge samples, achieving an average extraction efficiency of 81.9%. The studies determined that synthetic fibres were released in greater quantities than MPs over the short period during which the samples were collected. Recommendations for further methodological refinement are formulated and the need for authoritative methodological standardization is recognized. • Current understanding on the occurrence, fate and expulsion of MPs from WWTPs. • Assessment of analytical techniques used for MPs identification in samples of WWTPs. • Use of sustainable removal technologies for MPs in WWTPs. • Microplastic into renewable energy sources to facilitate the circular economy.

Sunlight-driven photocatalytic per- and polyfluoroalkyl substances degradation over zinc oxide/cellulose nanofiber catalyst using a continuous flow reactor

Recalcitrant pollutants in water with high resistance to natural degradation such as per/polyfluoroalkyl substances (PFAS) highlight the need for sustainable, cheap, and effective treatment approaches. Although photocatalysis under direct sunlight can be beneficial due to the usage of ambient conditions for the reaction, air as the oxidant, and sunlight as the energy source, identifying sustainable and sunlight photoactive materials and a process that is both scalable and industrially feasible are challenging. Herein, we report the use of ZnO/cellulose nanofiber (CNF) composites for the photodegradation of PFOA and PFOS upon irradiation by sunlight in a continuous flow photoreactor. HPLC/MS/MS and fluoride quantification using the SPADNS method were used to track the degradation of PFAS and by-product formation. Aqueous solutions of three different standard PFAS samples containing 1200 µg/L of PFOA, 800 µg/L PFOS, and a mixture of 900 µg/L of PFOA and 900 µg/L of PFOS, and a wastewater treatment plant sample with 2.5 µg/L of an environmental mixture of PFAS compounds present in urban wastewater were used for the photocatalytic degradation tests. The concentration of these pollutants and their by-products in these samples was reduced to 0.5 µg/L, 0.07 µg/L, 0.15 µg/L, and 0.3 µg/L with an EE/O figure of merit of 0.19, 0.28, 0.43, 0.88 kWh/m3 per order, respectively. It was observed that the standard mixture and the wastewater treatment plant samples were harder to degrade as compared to the standards containing one PFAS compound. Reusing the catalyst for three cycles showed less than 4% reduction in photodegradation over irradiation time. These findings emphasize the importance of location-specific design as the choice of photocatalyst is enabled by the UVA/B solar radiance characteristics present in Australia.

Experimental Protocol for Detecting SARS-CoV-2 in Screenings and Grit Samples of Wastewater Treatment Plants

Introduction: Although various liquid, solid, and gaseous streams of wastewater treatment plants (WWTPs) have been analyzed in many studies for the presence of SARS-CoV-2 RNA, no study was found to sample and detect SARS-CoV-2 RNA in screenings and grit samples separated from primary treatment units of WWTP. Hence, this study aims to provide an experimental protocol for sampling and extracting SARS-CoV-2 RNA from screenings and grits separated from WWTPs. Materials and Methods: First, sampling was conducted to extract SARS-CoV-2 RNA from screenings and grit samples. After sample processing and viral RNA extraction, SARS-CoV-2 RNA detection was performed by one-step reverse transcription quantitative polymerase chain reaction (RT-qPCR). Results: Based on the results, SARS-CoV-2 RNA was successfully extracted from screenings and grit samples of the studied WWTP with concentrations of (1.54 –3.9 × 104) and (0.8 – 2.3 × 104) genomic copies/L, respectively. Conclusion: Considering the successfully isolation and detection of SARS-CoV-2 viral RNA in solid phase samples of WWTP, this method can be applied for extracting SARS-CoV-2 RNA and maybe other viruses from the screenings and grit samples of WWTPs in related studies.

Application of a high-throughput quantitative PCR system for simultaneous monitoring of SARS-CoV-2 variants and other pathogenic viruses in wastewater

Variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are continuously emerging, highlighting the importance of regular surveillance of SARS-CoV-2 and other epidemiologically significant pathogenic viruses in the current context. Reverse transcription-quantitative PCR (RT-qPCR) is expensive, time-consuming, labor-intensive, requires a large reagent volume, and only tests a few targets in a single run. High-throughput qPCR (HT-qPCR) utilizing the Biomark HD system (Fluidigm) can be used as an alternative. This study applied an HT-qPCR to simultaneously detect SARS-CoV-2, SARS-CoV-2 nucleotide substituted RNA, and other pathogenic viruses in wastewater. Wastewater samples were collected from the coronavirus disease 2019 (COVID-19) quarantine facility between October 2020 and February 2021 (n = 4) and from the combined and separated sewer lines of a wastewater treatment plant (WWTP) in Yokkaichi, Mie Prefecture, Japan, between March and August 2021 (n = 23 each). The samples were analyzed by HT-qPCR using five SARS-CoV-2, nine SARS-CoV-2 spike gene nucleotide substitution-specific, five pathogenic viruses, and three process control assays. All samples from the quarantine facility tested positive for SARS-CoV-2 and the nucleotide substitutions N501Y and S69-70 del (Alpha variant) were detected in the December 2020 sample, coinciding with the first clinical case in Japan. Only three WWTP samples were positive when tested with a single SARS-CoV-2 assay, whereas more than eight samples were positive when tested with all assays, indicating that using multiple assays increases the likelihood of detection. The nucleotide substitution L452R (Delta variant) was detected in the WWTP samples of Mie Prefecture in April 2021, but the detection of Delta variant from patients had not been reported until May 2021. Aichi virus 1 and norovirus GII were prevalent in WWTP samples. This study demonstrated that HT-qPCR may be the most time- and cost-efficient method for tracking COVID-19 and broadly monitoring community health.

In-field one-step measurement of dissolved chemical oxygen demand with an integrated screen-printed electrochemical sensor

The increasing awareness of the importance of water for human and environmental health has prompted the need for developing fast, user-friendly and cost-effective analytical approaches for quality control and management. This work reports the fabrication and analytical assessment of a very simple sample-to-result electrochemical sensor for discrete and precise measurements of dissolved chemical oxygen demand (COD) in water samples of urban wastewater treatment plants (UWWTP). The device is fabricated by screen printing and shows a three-electrode cell configuration including a working electrode made of a composite material comprising a porous carbon matrix and copper nanoparticles, synthesized by a customized one-pot sol-gel process. The key feature of this device is the implementation of a paper disk loaded with sodium hydroxide covering the electrochemical cell area that allows sample filtering and pH conditioning. Thus, analytical measurements are carried out by casting a 20 µL sample drop without requiring any further user intervention. The overall sensor fabrication process can be scaled up for cost-effective mass manufacturing. The feasibility and performance of this integrated device are first evaluated using glucose as a standard analyte. A linear range up to 400 mg L− 1 O2 and a limit of detection of around 26 mg L− 1 O2 is achieved, this being well below the EU legal concentration limit of 125 mg L− 1 set for effluents of UWWTPs. Three real samples are analyzed in the lab as well as on-site and COD sensor values are in agreement with those obtained with the standard dichromate method at a certified laboratory, within the error limits. This miniaturized novel sensor platform greatly simplifies the standard analytical procedure for measuring COD, facilitating rapid decentralized analysis for precise control of urban wastewater treatment.

Chromatographic and spectroscopic comparison of dissolved organic matter variation in anaerobic-anoxic-oxic process with tertiary filtration and membrane bioreactor

Anaerobic-anoxic-oxic process with tertiary filtration (A2OWTF) and membrane bioreactor (MBR) was compared from the perspective of dissolved organic matter (DOM) variation in this study. A2OWTF and MBR wastewater treatment plants (WWTPs) samples were compared via conventional wastewater quality indexes, excitation-emission matrix (EEM) spectra, UV–vis spectra and high-performance size exclusion chromatography (HPSEC), which gradually revealed differences between the two processes from phenomena to mechanism. Lower removal of dissolved organic carbon (DOC) by MBR phenomenally revealed its DOM removal deficiency. EEM fluorescence regional integration (FRI), EEM parallel factor analysis (PARAFAC) and UV–vis Gaussian deconvolution qualitatively revealed more abundant humic-like substances (HS) and more complex effluent DOM composition for MBR. HPSEC provided more quantitative characterization. As for effluent after biotreatment, MBR exhibited lower removal rates of HPSEC fractions I (mainly containing protein and polysaccharides), IV (mainly containing low molecular weight organic acids) and fractions II, III (mainly containing HS) than A2OWTF (e.g., removal rates of MBR vs. A2OWTF calculated with UV absorbance at 254 nm (UVA254): 43.6% vs. 66.2%; 5.1% vs. 67.0%; −14.4% vs. 30.8%; −5.7% vs. 36.8% respectively for fractions I, IV, II, III). The massive HS generation in MBR also led to its higher trihalomethane formation potential (THMFP) for final effluent than A2OWTF. In conclusion, MBR should be prudently considered in terms of DOM removal for municipal WWTP upgrading.

Different fosA genes were found on mobile genetic elements in Escherichia coli from wastewaters of hospitals and municipals in Turkey

AimsThe increasing number of globally established fosfomycin-resistant (FosR) Gram-negative bacteria inspired us to investigate the occurrence of FosREnterobacterales populations (esp. E. coli) in samples of city wastewater treatment plants (WWTPs) and hospital sewage in Hatay, Turkey. FosR target bacteria were further characterized for their clonal relatedness, resistomes and mobile genetic elements (MGEs) to evaluate their impact on fosfomycin resistance dissemination. MethodsA total of 44 samples from raw and treated waters of WWTPs as well as of two hospitals in the Hatay province were subjected to selective cultivation for recovering FosREnterobacterales. The presence of fosA was verified by PCR and Sanger amplicon sequencing. Detected E. coli were further evaluated against antimicrobial susceptibility-testing, macrorestriction profiling (PFGE) and whole-genome sequencing (WGS). Bioinformatics analysis was performed for genome subtyping (i.e., MLST, serotype), resistome/virulome determination and dissection of the genetic determinants of plasmidic fosA3/4 resistances. ResultsBesides ten non-E. coli Enterobacterales, 29 E. coli were collected within this study. In silico-based subtyping revealed that E. coli isolates were assigned to six different serovars and 14 sequence types (ST), while O8:H21 and ST410 represented the major prevalent types, respectively. Fosfomycin resistance in the isolates was found to be mediated by the fosA4 (n = 18), fosA3 (n = 10) and fosA (n = 1), which are frequently associated with transmissible MGEs. Reconstruction of plasmid-associated fosA gene context revealed a linkage between the resistance cassette and IS6 (IS26 family) transposases, which might represent a major driver for the distribution of the genes and the generation of novel fosA-carrying plasmids. ConclusionsThe occurrence of plasmid-mediated, transmissible FosR in E. coli from wastewater pose a foreseeable threat to “One-Health”. To minimize further spread of the resistances in bacterial populations associated with environmental, animal and human health further resistance monitoring and management strategies must be developed.

Detection of SARS-CoV-2 RNA in wastewater, river water, and hospital wastewater of Nepal

The applicability of wastewater-based epidemiology (WBE) has been extensively studied throughout the world with remarkable findings. This study reports the presence and reduction of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) at two wastewater treatment plants (WWTPs) of Nepal, along with river water, hospital wastewater (HWW), and wastewater from sewer lines collected between July 2020 and February 2021. SARS-CoV-2 RNA was detected in 50%, 54%, 100%, and 100% of water samples from WWTPs, river hospitals, and sewer lines, respectively, by at least one of four quantitative PCR assays tested (CDC-N1, CDC-N2, NIID_2019-nCOV_N, and N_Sarbeco). The CDC-N2 assay detected SARS-CoV-2 RNA in the highest number of raw influent samples of both WWTPs. The highest concentration was observed for an influent sample of WWTP A (5.5 ± 1.0 log10 genome copies/L) by the N_Sarbeco assay. SARS-CoV-2 was detected in 47% (16/34) of the total treated effluents of WWTPs, indicating that biological treatments installed at the tested WWTPs are not enough to eliminate SARS-CoV-2 RNA. One influent sample was positive for N501Y mutation using the mutation-specific qPCR, highlighting a need for further typing of water samples to detect Variants of Concern. Furthermore, crAssphage-normalized SARS-CoV-2 RNA concentrations in raw wastewater did not show any significant association with the number of new coronavirus disease 2019 (COVID-19) cases in the whole district where the WWTPs were located, suggesting a need for further studies focusing on suitability of viral as well as biochemical markers as a population normalizing factor. Detection of SARS-CoV-2 RNA before, after, and during the peaking in number of COVID-19 cases suggests that WBE is a useful tool for COVID-19 case estimation in developing countries.

Tracking Klebsiella pneumoniae carbapenemase gene as an indicator of antimicrobial resistance dissemination from a hospital to surface water via a municipal wastewater treatment plant

Antibiotic-resistant bacteria originating from hospitals are ultimately discharged to municipal wastewater treatment plants (WWTP), which may serve as important reservoirs for the spread of antibiotic resistant genes. This study traced and quantified the presence of a rare but clinically relevant antimicrobial resistance gene; Klebsiella pneumoniae carbapenamase (KPC)-and the viable organisms (KPCO) which carried this gene in hospital, non-hospital wastewater discharges, various compartments within a municipal WWTP, receiving water and sediment samples. High concentration of the gene, blaKPC harbored in viable and multispecies KPCO was detected in the hospital wastewater and in the forepart stages of the WWTP, but was not detected in the final effluent following UV disinfection. KPCO were not detected in multiple non-hospital sources of wastewater discharges tested. The treatment train used in the sampled WWTP was found to help remove and reduce KPCO load. Using whole-genome sequencing, a KPC-producing Klebsiella oxytoca strain identical to strains seen in the patients and hospital environment was isolated from the downstream receiving water on one sampling event. KPCO were also found to persist in the biosolids throughout the WWTP, but were not detected in the processed compost-products made from WWTP-biosolids. This study systematically demonstrates dissemination of KPCO from hospital point source to environment via municipal WWTP. Understanding hospitals as the origin and source of spread of some of the most clinically urgent antimicrobial-resistant organisms may help direct interventions that target rate at which antibiotic resistant bacteria evolve and spread via enhancement of wastewater treatment and mitigation of dissemination at source.

A method to remove cellulose from rich organic samples to analyse microplastics

Knowing the amount of microplastics that currently reach wastewater is extremely important today. Furthermore, carrying out a good quantification and detection of the type of plastic provides valuable information. However, the wastewater is loaded, in addition to a high concentration of organic matter, with a high concentration of cellulose at the treatment plant influent, which seriously hinders detection, quantification and classification of microplastics. The abundance of cellulose materials makes them possible to become false positives for microplastics. Numerous studies on the analysis of microplastics in different matrices show how to remove organic matter from samples, but there are very few studies on the removal of cellulose, which is also found in the samples and hinders their analysis. This study offers a method that combines, for the analysis of microplastics, the already known advanced oxidation treatments for the elimination of organic matter with the novel cellulose removal treatment of the samples with the aim of reducing the amount of cellulose in the influent samples of conventional wastewater treatment plants. To remove the cellulose, 40 mL of a solution of urea 8%, sodium hydroxide 8% and thiourea 6.5% (by weight) were added for every 100 mg of dry sample. The beakers were placed in the freezer at minus 20 °C for 40 min and were then placed in agitation until they reached room temperature. After that, the samples were passed through a 53 μm mesh sieve. They were washed 15 times with 30 mL of ultra-pure water. The method is called UTS because of the acronym of its reagents (Urea/Thiourea/Sodium Hydroxide). By using the UTS method it is possible to reduce almost completely the cellulose residues from the influent sewage, and sludge samples by 97.6% in a second UTS treatment and 98.2% in a third UTS treatment. In all cases analyzed, the microplastics were identified as high density polyethylene (HDPE) with correlation indices higher than 0.97, which shows that the treatment is harmless for this type of plastic material. The UTS method in combination with the WPO is an efficient and effective method for the analysis of microplastics in different matrices where cellulose and organic matter may cause possible interferences.

Integrated efficiency of using nanocellulose-nano zero valent iron composite in water treatment

Water treatment using nano-materials can have a significant impact due to its surface properties. Coagulation techniques were studied by using 3 mL/L of 0.5% of different coagulants (polyacrylamide, poly aluminum chloride (PAC), ferric chloride, aluminum sulfate, and nanocellulose). Results indicated that the turbidity removal efficiency were 91.6%, 93.04%, 95.2%, 95.4%, and 99.4%, respectively. Treatment of water samples collected from the Ismailia Canal, the Damietta branch of the Nile Delta, and a wastewater treatment plant located in Cairo (Egypt) using nanocellulose fibers was studied. For the Ismailia Canal sample, the removal of turbidity, chemical oxygen demand (COD), biological oxygen demand (BOD), and phosphorous were 96%, 83.3%, 100%, and 100%, respectively. For the Damietta branch sample, the removal of turbidity, COD, BOD, and phosphorous were 87.5%, 81.3%, 88.9%, and 99.1%, respectively. For the wastewater treatment plant sample, the removal of turbidity, COD, BOD, and phosphorous were 86.4%, 91.96%, 92.86%, and 91.74%, respectively. Nanocellulose-nano zero-valent iron composite (NC-nZVI) was investigated for phosphorous removal at different operating conditions. Results showed phosphorous removal efficiencies of 91 and 100% for initial phosphate concentrations of 10 and 1 mg PO43– P/L, respectively. Different isothermal analyses were performed for monolayer and multilayer adsorption processes.

Rapid absolute quantification of pathogens and ARGs by nanopore sequencing

Compositional nature of relative abundance data in the current standard microbiome studies limits microbial dynamics interpretations and cross-sample comparisons. Here, we demonstrate the first rapid (1-h sequencing) method coupling Nanopore metagenomic sequencing with cellular spike-in to facilitate the absolute quantification and removal assessment of pathogens and antibiotic resistance genes (ARGs) in wastewater treatment plants (WWTPs). Nanopore sequencing-based quantification results for both simple mock community and complex real environmental samples showed a high consistency with those from the widely-used Illumina and culture-based approaches. Implementing such method, we quantified 46 predominant putative pathogenic species, and 361 ARGs in three WWTP sample sets. Though high log removals of dominant pathogens (2.23 logs) and ARGs (1.98 logs) were achieved, complete removal of all pathogens and ARGs were not achieved. Noticeably, Mycobacterium spp., Clostridium_P perfringens, and Borrelia hermsii exhibited low removal, and 13 ARGs even increased in absolute abundance after the treatment. Our proposed approach manifested its profound ability in providing absolute quantitation information guiding wastewater-based epidemiological surveillance and quantitative risk assessment facilitating microbial hazards management.

A nationwide indicator to smooth and normalize heterogeneous SARS-CoV-2 RNA data in wastewater

Since many infected people experience no or few symptoms, the SARS-CoV-2 epidemic is frequently monitored through massive virus testing of the population, an approach that may be biased and may be difficult to sustain in low-income countries. Since SARS-CoV-2 RNA can be detected in stool samples, quantifying SARS-CoV-2 genome by RT-qPCR in wastewater treatment plants (WWTPs) has been carried out as a complementary tool to monitor virus circulation among human populations. However, measuring SARS-CoV-2 viral load in WWTPs can be affected by many experimental and environmental factors. To circumvent these limits, we propose here a novel indicator, the wastewater indicator (WWI), that partly reduces and corrects the noise associated with the SARS-CoV-2 genome quantification in wastewater (average noise reduction of 19%). All data processing results in an average correlation gain of 18% with the incidence rate. The WWI can take into account the censorship linked to the limit of quantification (LOQ), allows the automatic detection of outliers to be integrated into the smoothing algorithm, estimates the average measurement error committed on the samples and proposes a solution for inter-laboratory normalization in the absence of inter-laboratory assays (ILA). This method has been successfully applied in the context of Obépine, a French national network that has been quantifying SARS-CoV-2 genome in a representative sample of French WWTPs since March 5th 2020. By August 26th, 2021, 168 WWTPs were monitored in the French metropolitan and overseas territories of France. We detail the process of elaboration of this indicator, show that it is strongly correlated to the incidence rate and that the optimal time lag between these two signals is only a few days, making our indicator an efficient complement to the incidence rate. This alternative approach may be especially important to evaluate SARS-CoV-2 dynamics in human populations when the testing rate is low.

On-line solid-phase extraction of pharmaceutical compounds from wastewater treatment plant samples using restricted access media in column-switching liquid chromatography-tandem mass spectrometry

An on-line solid phase extraction using a lab-made restricted access media (RAM) was developed as sample preparation procedure for determination of the pharmaceutical compounds caffeine (CAF), carbamazepine (CBZ), norfloxacin (NOR), ciprofloxacin (CIP), fluoxetine (FLX) and venlafaxine in wastewater treatment plant samples by liquid chromatography-tandem mass spectrometry (LC-MS/MS). This method is suitable for use in routine of analysis, avoiding cross-contamination and requiring only a small sample volume (50 µL), with minimal handling.The method was validated according to international guidelines. The chromatographic efficiency was evaluated using peak resolution and asymmetry parameters. Carryover was also evaluated, in order to ensure reliability of the analysis and the ability to reuse the cartridge. Satisfactory linearity (r2 > 0.99) was obtained for all the compounds. The intra- and inter-day precision values were lower than 5.79 and 14.1%, respectively. The limits of detection ranged from 0.01 to 3 µg L−1 and the limits of quantification were from 0.1 to 5 µg L−1. The method was applied to 20 environmental wastewater samples, with caffeine being the most widely detected compound, at the highest concentration of 392 µg L−1, while other compounds were detected in fewer samples at lower concentrations (up to 9.60 µg L−1). The lab-made modification is a cheaper option for on-line sample preparation, compared to commercially available on-line SPE cartridges and RAM columns. Moreover, a high-throughput procedure was achieved, with an analysis time of 16 min including sample preparation and chromatographic separation. The same RAM column was applied over 200 injections including method optimization, validation and application in wastewater samples without loss of analytical response.

Formation of transformation products during ozonation of secondary wastewater effluent and their fate in post-treatment: From laboratory- to full-scale

Ozonation is increasingly applied in water and wastewater treatment for the abatement of micropollutants (MPs). However, the transformation products formed during ozonation (OTPs) and their fate in biological or sorptive post-treatments is largely unknown. In this project, a high-throughput approach, combining laboratory ozonation experiments and detection by liquid chromatography high-resolution mass spectrometry (LC-HR-MS/MS), was developed and applied to identify OTPs formed during ozonation of wastewater effluent for a large number of relevant MPs (total 87). For the laboratory ozonation experiments, a simplified experimental solution, consisting of surrogate organic matter (methanol and acetate), was created, which produced ozonation conditions similar to realistic conditions in terms of ozone and hydroxyl radical exposures. The 87 selected parent MPs were divided into 19 mixtures, which enabled the identification of OTPs with an optimized number of experiments. The following two approaches were considered to identify OTPs. (1) A screening of LC-HR-MS signal formation in these experiments was performed and revealed a list of 1749 potential OTP candidate signals associated to 70 parent MPs. This list can be used in future suspect screening studies. (2) A screening was performed for signals that were formed in both batch experiments and in samples of wastewater treatment plants (WWTPs). This second approach was ultimately more time-efficient and was applied to four different WWTPs with ozonation (specific ozone doses in the range 0.23-0.55 gO3/gDOC), leading to the identification of 84 relevant OTPs of 40 parent MPs in wastewater effluent. Chemical structures could be proposed for 83 OTPs through the interpretation of MS/MS spectra and expert knowledge in ozone chemistry. Forty-eight OTPs (58%) have not been reported previously. The fate of the verified OTPs was studied in different post-treatment steps. During sand filtration, 87-89% of the OTPs were stable. In granular activated carbon (GAC) filters, OTPs were abated with decreasing efficiency with increasing run times of the filters. For example, in a GAC filter with 16,000 bed volumes, 53% of the OTPs were abated, while in a GAC filter with 35,000 bed volumes, 40% of the OTPs were abated. The highest abatement (87% of OTPs) was observed when 13 mg/L powdered activated carbon (PAC) was dosed onto a sand filter.

Eco-efficiency evaluation in wastewater treatment plants considering greenhouse gas emissions through the data envelopment analysis-tolerance model

The eco-efficiency evaluation in wastewater treatment plants (WWTPs) is used to know and improve the environmental and economic efficiency of these processes, systems, products, and services. The eco-efficiency evaluations in WWTP contemplate the inputs to be minimized, the desirable results to be maximized, and the undesired results to be minimized. Data envelopment analysis (DEA) is a widely used method to evaluate the eco-efficiency of WWTPs; integrating several approaches in a single index, traditional DEA models do not take into account the uncertainty in the data. This study evaluates the eco-efficiency of a sample of Catalan WWTPs, considering the uncertainty of the data (DEA tolerance model), and it is for the first time that together with CO2, other greenhouse gas (GHG) such as CH4 and N2O are considered as part of the process outputs. GHG emissions were quantified using methods reported in the literature. Seven hundred twenty-nine eco-efficiency scores were estimated for each WWTP instead of a single score like conventional DEA models, analyzing optimistic and pessimistic scenarios. The WWTPs were classified according to the estimated eco-efficiency scores, accounting for the uncertainty in each of the scenarios, and demonstrating the changes in the performance of the WWTPs in the different scenarios. Only two WWTPs were eco-efficient in all the scenarios evaluated. This approach provides essential information to improve efficiency and innovation in the wastewater sector.

Characterisation of (anti-)progestogenic and (anti-)androgenic activities in surface and wastewater using high resolution effectdirected analysis

The quality of surface waters is threatened by pollution with low concentrations of bioactive chemicals, among which those interfering with steroid hormone systems. Induced by reports of anti-progestogenic activity in surface waters, a two-year four-weekly survey of (anti-)progestogenic activity was performed at three surface water locations in the Netherlands that serve as abstraction points for the production of drinking water. As certain endogenous and synthetic progestogenic compounds are also potent (anti-)androgens, these activities were also investigated. Anti-progestogenic and anti-androgenic activities were detected in the majority of the monitoring samples, sometimes in concentrations exceeding effect-based trigger values, indicating the need for further research. To characterize the compounds responsible for the activities, a high resolution Effect-Directed Analysis (hr-EDA) panel was combined with PR and AR CALUX bioassays, performed in agonistic and antagonistic modes. The influent and effluent of a domestic wastewater treatment plant (WWTP) were included as effluent is a possible emission source of active compounds. As drivers for androgenic and progestogenic activities several native and synthetic steroid hormones were identified in the WWTP samples, namely androstenedione, testosterone, DHT, levonorgestrel and cyproterone acetate. The pesticides metolachlor and cyazofamid were identified as contributors to both the anti-progestogenic and anti-androgenic activities in surface water. In addition, epiconazole contributed to the anti-progestogenic activities in the rivers Rhine and Enclosed Meuse. This study showed the strength of hr-EDA for the identification of bioactive compounds in environmental samples and shed light on the drivers of (anti-)progestogenic and (anti-)androgenic activities in the aquatic environment.