Non-target liquid chromatography high-resolution mass spectrometry screening to prioritize unregulated micropollutants that persist through domestic wastewater treatment.

Efforts to regulate and monitor emerging contaminants are insufficient because new chemicals are continually brought to market, and many are unregulated and potentially harmful. Domestic wastewater treatment plants are not designed to remove micropollutants and are important sources of emerging contaminants in the aquatic environment. In this study, non-target screening, an unbiased method for analyzing compounds without prior information, was used to identify compounds that may be emitted in wastewater treatment plant effluent and should be monitored. Nine wastewater treatment plants using different treatment methods were studied, and a non-target screening data-processing method was used. The frequencies at which the contaminants were detected and contaminant persistence through the treatment processes were considered, and then the contaminants were prioritized. The predicted no-effect concentration of each prioritized contaminant was used to determine whether further analysis and monitoring of the contaminant was necessary. Quantitative analyses of five compounds (amantadine, atenolol, benzotriazole, diphenhydramine, and sulpiride) were performed using reference standards. Probable molecular formulae and structures were proposed for 17 contaminants, and the risks posed by the contaminants were estimated using predicted no-effect concentrations. The results provide valuable insights into how unregulated micropollutants can be identified and prioritized for monitoring in future studies.

Impact of chlorine disinfection on intracellular and extracellular antimicrobial resistance genes in wastewater treatment and water reclamation.

Wastewater treatment plants and water reclamation facilities are reservoirs of antimicrobial resistance genes (ARGs). These ARGs are not limited solely to intracellular DNA (inARGs) but include extracellular DNA (exARGs) present in wastewater. The release of exARGs from cells can be exacerbated by treatment processes, including chlorine disinfection, which disrupts bacterial cells. Given the potential for exARGs to drive horizontal gene transfer and contribute to the proliferation of antimicrobial resistance, it is imperative to recognize these fractions as emerging environmental pollutants. In this study, we conducted a comprehensive year-long assessment of both inARGs and exARGs, further differentiating between dissolved exARGs (Dis_exARGs) and exARGs adsorbed onto particulate matter (Ads_exARGs), within a full-scale wastewater treatment and water reclamation facility. The results revealed that Ads_exARGs comprised up to 30 % of the total ARGs in raw sewage with high biomass content. Generally, treatments at low and high doses of chlorine increased the abundance of Dis_exARGs and Ads_exARGs. The fate of ARG levels that varied depending on the type of ARGs suggested variations in the susceptibility of the host bacteria to chlorination. Moreover, co-occurrence of several potential opportunistic pathogenic bacteria and ARGs were observed. Therefore, we propose higher doses of chlorination as a prerequisite for the effective removal of inARGs and exARGs.

Sensitivity and quantitativeness of large-volume injection combined with liquid chromatography/high-resolution mass spectrometry for target screening analysis of emerging contaminants.

Solid phase extraction (SPE) has been widely used for pretreatment in target screening (TS) analysis. However, some compounds are difficult to recover by SPE or their recovery is unstable for environmental samples. In this study, we tested large-volume injection (LVI) without SPE for TS analysis of 103 compounds listed by the Ministry of the Environment (Japan)-so-called 'items to be surveyed'-using liquid chromatography high-resolution mass spectrometry. We evaluated the limit of quantification (LOQ) by LVI and compared this LOQ with the LOQ by SPE pretreatment using a hydrophilic-lipophilic balance (HLB) combined with activated carbon, which was found previously to afford the best SPE cartridges for target compounds recovery. The LOQ generally decreased as the injection volume increased, and the LOQ was at least 250 times lower for a 500-µL injection than for a 2-µL injection for half of the compounds. LVI provided LOQs lower than the predicted no effect concentration for more compounds than the SPE method. The average matrix effect (ME) by LVI was in the range 70%-130% for 69 out of 97 compounds. The ME was higher or lower for some of the remaining compounds, but the ME was in the range 10%-1000% for all 18 water samples for 84 of the 97 compounds. Comparing the ME by LVI and the recovery ratio by the SPE method showed that LVI achieved more accurate quantitation than the SPE method for a larger number of compounds. Therefore, LVI provides better sensitivity and quantitativeness than the SPE method using HLB and activated carbon for TS analysis of as many 'items to be surveyed' as possible.

Autotrophic growth activity of complete ammonia oxidizers in an upflow biological contact filter for drinking water treatment.

Biological filters effectively remove ammonium from drinking water via nitrification. In a pilot-scale upflow biological contact filter (U-BCF), complete ammonia oxidizers (comammox), which are capable of oxidizing ammonia to nitrate in one cell, were more abundant than ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB). However, little information is available on the contribution of comammox to nitrification. In this study, we evaluated the autotrophic growth activity of comammox associated with biological activated carbon (BAC) in a U-BCF by DNA-stable isotope probing (DNA-SIP). BAC samples collected from the U-BCF were continuously fed mineral medium containing 0.14 mg N L-1 ammonium and 12C- or 13C-labeled bicarbonate for 20 days. DNA-SIP analysis revealed that comammox (clades A and B) as well as AOA assimilated bicarbonate after 10 days of incubation, proving that dominant comammox could contribute to nitrification. Contrarily, AOB remained inactive throughout the observation period. Amplicon sequencing of the 13C-labeled DNA fractions of comammox revealed that specific genotypes other than the most dominant genotype in the original sample were more enriched under the incubation condition for the DNA-SIP experiment. Thus, dominant genotypes of comammox in a U-BCF might utilize organic nitrogen to fuel nitrification in ammonia-limited environments.

Dynamics of the Microbial Community and Opportunistic Pathogens after Water Stagnation in the Premise Plumbing of a Building.

In premise plumbing, microbial water quality may deteriorate under certain conditions, such as stagnation. Stagnation results in a loss of disinfectant residual, which may lead to the regrowth of microorganisms, including opportunistic pathogens. In the present study, microbial regrowth was investigated at eight faucets in a building over four seasons in one year. Water samples were obtained before and after 24 h of stagnation. In the first 100| |mL after stagnation, total cell counts measured by flow cytometry increased 14- to 220-fold with a simultaneous decrease in free chlorine from 0.17-0.36| |mg L-1 to <0.02| |mg L-1. After stagnation, total cell counts were not significantly different among seasons; however, the composition of the microbial community varied seasonally. The relative abundance of Pseudomonas spp. was dominant in winter, whereas Sphingomonas spp. were dominant in most faucets after stagnation in other seasons. Opportunistic pathogens, such as Legionella pneumophila, Mycobacterium avium, Pseudomonas aeruginosa, and Acanthamoeba spp., were below the quantification limit for real-time quantitative PCR in all samples. However, sequences related to other opportunistic pathogens, including L. feeleii, L. maceachernii, L. micdadei, M. paragordonae, M. gordonae, and M. haemophilum, were detected. These results indicate that health risks may increase after stagnation due to the regrowth of opportunistic pathogens.

Emergence of mobile tigecycline resistance gene tet(X4)-harbouring Shewanella xiamenensis in a water environment.

OBJECTIVES: Tigecycline resistance mediated by the mobile tigecycline-inactivating enzyme gene tet(X) in Gram-negative bacteria is an emerging concern for global public health. However, limited information is available on the distribution of tet(X) in the natural environment. In this study, we investigated the presence of tet(X) in environmental Gram-negative bacteria. METHODS: A carbapenem- and tigecycline-resistant Shewanella xiamenensis isolate (NUITM-VS1) was obtained from an urban drainage in Hanoi, Vietnam, in March 2021. Whole-genome sequencing analysis was performed by long- and short-read sequencing, resulting in a complete genome sequence. Antimicrobial resistance genes (ARGs) in the genome were detected based on the custom ARG database, including all known tigecycline resistance genes. RESULTS: Shewanella xiamenensis isolate NUITM-VS1 harboured the tet(X4) gene and the blaOXA-48 carbapenemase gene on the chromosome. tet(X4) was flanked by IS91 family transposase genes, suggesting that the acquisition of tet(X4) was mediated by this mobile gene element (MGE), whereas no MGE was found surrounding blaOXA-48, consistent with previous findings that blaOXA-48-like ß-lactamase genes are species-specific intrinsic ARGs in Shewanella spp. CONCLUSION: To the best of our knowledge, this is the first report of a tet(X4)-harbouring Shewanella sp. isolate. Our results provide genetic evidence of the complexity of the dynamics of clinically important ARGs among bacteria in the water environment.

A Transferable IncC-IncX3 Hybrid Plasmid Cocarrying blaNDM-4, tet(X), and tmexCD3-toprJ3 Confers Resistance to Carbapenem and Tigecycline.

Tigecycline is a last-resort antimicrobial against carbapenemase-producing Enterobacterales (CPE). However, mobile tigecycline resistance genes, tet(X) and tmexCD-toprJ, have emerged in China and have spread possibly worldwide. Tet(X) family proteins function as tigecycline-inactivating enzymes, and TMexCD-TOprJ complexes function as efflux pumps for tigecycline. Here, to the best of our knowledge we report a CPE isolate harboring both emerging tigecycline resistance factors for the first time. A carbapenem- and tigecycline-resistant Klebsiella aerogenes strain, NUITM-VK5, was isolated from an urban drainage in Vietnam in 2021, and a plasmid, pNUITM-VK5_mdr, cocarrying tet(X) and tmexCD3-toprJ3 along with the carbapenemase gene blaNDM-4 was identified in NUITM-VK5. pNUITM-VK5_mdr was transferred to Escherichia coli by conjugation and simultaneously conferred high-level resistance against multiple antimicrobials, including carbapenems and tigecycline. An efflux pump inhibitor reduced TMexCD3-TOprJ3-mediated tigecycline resistance, suggesting that both tigecycline resistance factors independently and additively contribute to the high-level resistance. The plasmid had the IncX3 and IncC replicons and was estimated to be a hybrid of plasmids with different backbones. Unlike IncX3 plasmids, IncC plasmids are stably maintained in an extremely broad range of bacterial hosts in humans, animals, and the environment. Thus, the future global spread of multidrug resistance plasmids such as pNUITM-VK5_mdr poses a public health crisis. IMPORTANCE Tigecycline is important as a last-resort antimicrobial and effective against antimicrobial-resistant bacteria, such as carbapenem-producing Enterobacterales (CPE), whose infections are difficult to treat with antimicrobials. Since 2019, mobile tigecycline resistance genes, tet(X) and tmexCD-toprJ, and their variants have been reported mainly from China, and it has become important to understand their epidemiological situation and detailed genetic mechanisms. In this study, we identified a bacterial isolate coharboring tet(X) and tmexCD-toprJ on the same plasmid. A Klebsiella aerogenes isolate in Vietnam carried both these tigecycline resistance genes on a transferable plasmid leading to high-level resistance to multiple clinically important antimicrobials, including carbapenem and tigecycline, and could actually transfer the plasmid to other bacteria. The spread of such a multidrug resistance plasmid among bacterial pathogens should be of great concern because there are few antimicrobials to combat bacteria that have acquired the plasmid.

Identification of new eligible indicator organisms for combined sewer overflow via 16S rRNA gene amplicon sequencing in Kanda River, Tokyo.

Fecal indicator bacteria (FIB) are commonly used to evaluate the pollution impact of combined sewer overflows (CSOs) in urban rivers. Although water quality assessment with FIB has a long tradition, recent studies demonstrated that FIB have a low correlation with pathogens and therefore are not accurate enough for the assessment of potential human hazards in water. Consequently, new eligible and more specific indicators have to be identified, which was done in this study via sequencing of genetic markers from total community DNA. To identify potential microbiome-based indicators, microbial communities in samples from an urban river in Tokyo under different climatic conditions (dry and rainy) were compared with the influent and effluent of three domestic wastewater treatment plants (WWTPs) by analyzing 16 S rRNA gene amplicon libraries. In the first part of this study, physicochemical parameters and FIB quantification with selective culture techniques facilitated the identification of samples contaminated with CSO, sewage, or both. This allowed the grouping of samples into CSO-contaminated and non-contaminated samples, an essential step prior to the microbiome comparison between samples. Increased turbidity, ammonia concentrations, and E. coli [up to (9.37 ± 0.95) × 102 CFU/mL after 11.5 mm of rainfall] were observed in CSO-contaminated river samples. Comparison of dry weather (including WWTP samples) and rainy weather samples showed a reduction in microbial diversity in CSO-contaminated samples. Furthermore, the results of this study suggest Bacteroides spp. as a novel indicator of sewage pollution in surface waters.

Non-target screening of dissolved organic matter in raw water, coagulated water, and chlorinated water by Orbitrap mass spectrometry.

This study aimed to classify the possible molecular formulas of precursors for disinfection by-products (DBPs) in raw, coagulated, and chlorinated water samples from the U-Tapao Canal, Songkhla, Thailand. The molecular formulas of DBPs in chlorinated water were investigated. Polyaluminum chloride (PACl) was employed as a coagulant. Orbitrap Fourier transform-mass spectrometry was able to estimate the composition of dissolved organic matter (DOM) with the carbon (C), hydrogen (H), oxygen (O), nitrogen (N), and sulfur (S) elements contained and DBPs at the molecular level. The molecular formulas of the DOM in the raw water primarily consisted of CHO and CHON when extracted by lichrolut EN. The CHO and CHON species were rich in lignin-, tannin-, and condensed aromatic-like substances. The DOM with high-molecular-weight from 300 to 500 Da were preferentially removed by coagulation. The PACl coagulation decreased the abundances of lignin-, tannin-, and condensed aromatic-like substances in the CHO formulas, while lignin- and condensed aromatic-like substances in the CHON formulas remained. The remaining precursors corresponded to CHON molecules in the coagulated water, which may result in the formation of some chlorine (Cl)-containing molecules. Several DBPs among the CHOCl and CHONCl species were produced in the chlorinated water through the addition reaction of chlorine. New chlorinated N-DBPs of 21 formulas were detected.

Changes in dissolved organic matter during water treatment by sequential solid-phase extraction and unknown screening analysis.

Isolation of complex dissolved organic matter (DOM) from environmental water is a major challenge for unknown screening analysis by high-resolution mass spectrometry. In this study, DOM in process water during advanced drinking water treatment was fractionated sequentially by three solid-phase extraction (SPE) cartridges based on the polarity and charge of DOM molecules. By sequential SPE with unknown screening analysis, over 3000 DOM features were found in raw water, whereas around 2000 were obtained by a single SPE. The hydrophobic neutral (HPON) fraction contained CHO features with highest averaged molecular weight followed by hydrophobic acid (HPOA) and then hydrophilic acid (HPIA). The average degree of carbon double bond equivalents and carbon oxidation states indicated that the HPON fraction contained molecules that were more unsaturated and less oxidized than those of the HPOA and HPIA fractions. Ozone selectively decomposed (1) more unsaturated and less oxidized HPON features, (2) more unsaturated HPOA compounds, and (3) less oxidized HPIA molecules. Oxidation by-products were mostly HPON and HPIA compounds that were more oxidized than the decomposed molecules. During biological activated carbon (BAC) filtration, less oxidized HPON were preferentially removed, whereas HPOA were removed without selectivity. HPON and HPIA molecules with more oxidized character were found to be refractory to BAC treatment. HPON with more unsaturated and HPIA with more oxidized characters were decomposed by chlorine. Many types of HPIA decomposed during chlorination were the oxidation by-products of ozonation that were refractory to BAC treatment. Sequential SPE with unknown screening analysis provided previously unknown details of the molecular characteristics of DOM and its changes during advanced water treatment.

Temperature-Dependent Ammonium Removal Capacity of Biological Activated Carbon Used in a Full-Scale Drinking Water Treatment Plant.

Nitrification is a key function of biological activated carbon (BAC) filters for drinking water treatment. It is empirically known that the nitrification activity of BAC filters depends on water temperature, potentially resulting in the leakage of ammonium from BAC filters when the water temperature decreases. However, the ammonium removal capacity of BAC filters and factors governing the capacity remain unknown. This study employed a bench-scale column assay to determine the volumetric ammonium removal rate (VARR) of BAC collected from a full-scale drinking water treatment plant. VARR was determined at a fixed loading rate under different conditions. Seasonal variations of the VARR as well as impacts of the water matrix and water temperature on ammonium removal were quantitatively analyzed. While the VARR in an inorganic medium at 25 °C was maintained even during low water temperature periods and during breakpoint chlorination periods, the water matrix factor reduced the VARR in ozonated water at 25 °C by 33% on average. The VARR in ozonated water was dependent on water temperature, indicating that the microbial activity of BAC did not adapt to low water temperature. The Arrhenius equation was applied to reveal the relationship between VARR and water temperature. The actual ammonium removal performance of a full-scale BAC filter was predicted. VARR is useful for water engineers to reexamine the loading and filter depth of BAC filters.

Whole-genome sequencing and comparative genomic analysis of Shewanella xiamenensis strains carrying blaOXA-48-like genes isolated from a water environment in Vietnam.

OBJECTIVES: The aim of this study was to understand the natural bacterial hosts of antimicrobial resistance genes (ARGs) and their impact on the processes of evolution, spread and positive selection of acquired ARGs. METHODS: Environmental carbapenem-resistant Gram-negative bacteria in Vietnam were screened for based on a One Health approach. Whole-genome sequencing (WGS) and comparative genomic analysis of the isolates were performed. WGS of three carbapenem-resistant Shewanella xiamenensis strains (SxND_W2_2018, SxND_W5_2018 and SxND_W9_2018) isolated from canals in Truc Ninh District and Nghia Hung District, Nam Dinh Province, Vietnam, in 2018 was performed using an Illumina MiniSeq system. ARGs in the draft genome sequences were detected using ResFinder, and comparison of genomic regions was performed using BLASTn and Easyfig. RESULTS: TheblaOXA-48-like carbapenem-hydrolysing class D ß-lactamase genes blaOXA-48, blaOXA-252 and blaOXA-547 were detected in chromosomal contigs of SxND_W2_2018, SxND_W5_2018 and SxND_W9_2018, respectively. Comparative analysis of the surrounding regions of the blaOXA-48-like genes, including both 10 kb upstream and 10 kb downstream of the genes, showed that the genomic regions were highly conserved in all three isolates. CONCLUSION: This study analysed the draft genome sequences of carbapenem-resistantS. xiamenensis strains isolated from a water environment in Vietnam. All of the strains carried blaOXA-48-like gene variants in their chromosomes. This information will contribute to highlight the evolution of blaOXA-48 family carbapenemase genes in nature and the importance of S. xiamenensis as a natural reservoir of important ARGs in the environment in Vietnam.

Metal partitioning and leaching vulnerability in soil, soakaway sediments, and road dust in the urban area of Japan.

Isotope dilution techniques (IDT) and sequential extraction procedures (SEPs) were compared to apprehend the differences between two techniques in determining metal exchangeability and vulnerability to pollute the urban groundwater. For this purpose, soil (n = 2), "soakaway" sediment deposited in the artificial infiltration facilities (AIF) (n = 4), and road dust (n = 2) were sampled from Tokyo metropolitan. Sorption coefficients of four metals (Cu, Zn, Cd and Pb) were assessed through isotopic exchangeability (E-value) and potential mobile pool i.e. addition of exchangeable, reducible and oxidizable fraction obtained by Community Bureau of Reference (BCR)-procedures. The E-value for the three samples were found smaller than the potential mobile pool but were higher than BCR-exchangeable fractions. The use of strong extractants are likely to play an active role in the disagreement between SEPs and IDT. IDT accounts for the isotopic exchangeability while BCR provides information of vulnerability of metals associated with different fractions that can leach under different environmental conditions. Sorption coefficients measured in soakaway sediment was found comparable to soil thus likely to retain metals. However, as variability in environmental conditions is likely to affect Kd, the soakaway sediment may become an active metal source in future rather than acting as the permanent sink. The study concludes that there is the possibility of errors while predicting metal vulnerability to groundwater with both techniques and thus a model compliance integrating the virtue of both techniques will be a way forward.

Consecutive ultrafiltration and silica adsorption for recovery of extracellular antibiotic resistance genes from an urban river.

The dissemination of antibiotic resistance (AR) has attracted global attention because of the increasing antibiotic treatment failure it has caused. Through natural transformation, a live bacterium takes up extracellular DNA (exDNA), which facilitates AR dissemination. However, recovery of exDNA from water samples is challenging. In this study, we validated a consecutive ultrafiltration-based protocol to simultaneously recover intracellular DNA (inDNA), dissolved exDNA (Dis_exDNA, dissolved in the bulk water), and adsorbed exDNA (Ads_exDNA, adsorbed to the surfaces of suspended particles). Using hollow fiber ultrafiltration (HFUF), all DNA fractions were concentrated from environmental water samples, after which Dis_exDNA (supernatant) was separated from inDNA and Ads_exDNA (pellets) using centrifugation. Ads_exDNA was washed off from the pellets with proteinase K and sodium phosphate buffer. Dis_exDNA and Ads_exDNA were further concentrated using centrifugal ultrafiltration, from which silica binding was performed. inDNA was extracted from washed pellets with a commercial kit. For inDNA, HFUF showed recovery efficiencies of 96.5 ± 18.5% and 88.0 ± 2.0% for total cells and cultured Escherichia coli, respectively (n = 3). To represent all possible DNA fragments in water environment, exDNA with different lengths (10.0, 4.0, 1.0, and 0.5 kbp) were spiked to test the recovery efficiencies for Dis_exDNA. The whole process achieved 62.2%-62.9% recovery for 10 and 4 kbp exDNA, and 38.8%-44.5% recovery for 1.0 and 0.5 kbp exDNA. Proteinase K treatment enhanced the recovery of Ads_exDNA by 4.0-10.7 times. The protocol was applied to water samples from an urban river in Tokyo, Japan. The abundance of AR genes (ARGs) in inDNA, Dis_exDNA, and Ads_exDNA increased downstream of wastewater treatment plants. ARGs in Ads_exDNA and Dis_exDNA accounted for 1.8%-26.7% and 0.03%-20.9%, respectively, of the total DNA, implying that Ads_exDNA and Dis_exDNA are nonnegligible potential pools for the horizontal transfer of ARGs.

Simultaneous screening for chemically diverse micropollutants in public water bodies in Japan by high-performance liquid chromatography-Orbitrap mass spectrometry.

An improved assessment of environmental risks to public water bodies requires screening a large number of micropollutants. This study reports the development of a novel target screening method based on solid-phase extraction (SPE), HPLC, and high-resolution Orbitrap MS for the analysis of micropollutants with diverse chemical properties. First, target compounds were screened for their detectability by Orbitrap MS. An optimized SPE cartridge and HPLC column maximized recovery and separated most target compounds. The sensitivity and repeatability of the method was validated by determining the detection limits and relative standard deviation (RSD). Eighty-four compounds with highly diverse properties were simultaneously detected with detection limits of 0.1-100 ng/L. Of these compounds, 52 were quantitated, with R2 ≥ 0.99 by linearity analysis and SPE recovery ratios of ≥50%. The remaining 32 compounds were qualitatively detected, with R2 < 0.99 or SPE recovery ratio of <50%. Satisfactory repeatability was obtained (RSD < 13.5%). This method was applied to the surveillance of the Arakawa River in Japan in 2019. Thirty-two compounds, including pesticides, surfactants, plasticizers, adhesives, and industrial solvents, were detected in the river. The measured concentrations of 13 compounds were compared with their predicted no effect concentrations (PNECs). Decanoic acid showed a higher concentration than the corresponding PNEC value, suggesting that its risk to the Arakawa water environment required further evaluation. The concentrations of dicyclohexylamine, 1,3-diphenylguanidine, and 2,4-dichlorophenoxyacetic acid were higher than their corresponding PNEC/10 values, demonstrating that these compounds were of higher priority than other compounds.

Molecular characteristics of dissolved organic matter transformed by O3 and O3/H2O2 treatments and the effects on formation of unknown disinfection by-products.

We investigated semiquantitative changes in almost 1000 dissolved organic matter (DOM) features during oxidation with 1 mg of O3 per liter (mg O3/L), 4 mg O3/L, or 4 mg O3/L + 2.5 mg of H2O2 per liter (advanced oxidation process, AOP) by unknown screening analysis with Orbitrap mass spectrometry. The consequential effects on formation of unknown disinfection by-products (DBPs) by chlorination were evaluated in laboratory-scale experiments. Several hundred unsaturated DOM features with positive oxygen-subtracted double bond equivalents per carbon ((DBE-O)/C) were decomposed by the ozone-only treatment and AOP. The AOP decomposed some saturated (negative (DBE-O)/C)) and reduced molecules, which had negative carbon oxidation states (Cos). Several hundred saturated oxidation by-products were detected after ozonation and the AOP. After chlorination, the samples pre-treated with ozone alone resulted in higher formation of unknown DBPs than the AOP pre-treated sample or the sample without oxidation. Over half of the DBP precursors, estimated by electrophilic substitution, were not totally decomposed by any oxidation process, but they were increased after the ozone-only process and AOP. DBP precursors produced by the ozone-only process or AOP formed unique unknown DBPs. Therefore, post-treatment processes after oxidation and before chlorination are important to minimize formation of unknown DBPs.

Viability RT-qPCR Combined with Sodium Deoxycholate Pre-treatment for Selective Quantification of Infectious Viruses in Drinking Water Samples.

The presence of pathogenic viruses in drinking water is a major public health concern. Although viability RT-qPCR methods were developed to quantify infectious viruses, they may not always reflect viral infectivity, therefore leading to false-positive results. In this study, sodium deoxycholate (SD) pre-treatment was used to improve the efficiency of viability RT-qPCR methods with respect to exclusive quantification of infectious viruses. The ability of SD pre-treatment to enhance the penetration of three viability markers, namely, ethidium monoazide (EMA, 100 µM), propidium monoazide (PMA, 100 µM), and cis-dichlorodiammineplatinum (CDDP, 1000 µM), into heat-treated (90 °C for 1 min) Aichi virus at various concentrations (0.01-0.5%) was evaluated. The optimal SD concentration was found to be 0.1% for all markers. EMA/PMA/CDDP-RT-qPCR with 0.1% SD pre-treatment was significantly more effective than without SD pre-treatment in determining AiV inactivation after heat (50, 60, 70, 80, or 90 °C for 1 min) or chlorine treatment (1 mgCl2/L for 1, 2, 5, or 10 min). Among the viability RT-qPCR methods tested, CDDP-RT-qPCR with SD pre-treatment (SD-CDDP-RT-qPCR) was the most effective in reflecting viral infectivity. Performance testing of SD-CDDP-RT-qPCR in concentrated drinking water samples did not reveal any significant effects of SD-CDDP treatment. Thus, SD-CDDP-RT-qPCR could be a useful tool for monitoring infectious virus presence in drinking water.

Ferrihydrite treatment to mitigate inhibition of RT-qPCR virus detection from large-volume environmental water samples.

Molecular assays are currently the most widely used method to quantify pathogenic viruses in water; however, their performance is often disrupted by matrices present in environmental samples. The present study used ferrihydrite (Fh) treatment to mitigate inhibition of RT-qPCR virus detection from environmental water concentrates. Fh treatment was performed to improve the detection of spiked Aichi virus 1 (AiV) and Qß bacteriophage (Qß) in commercial humic acids and seawater concentrates. The optimal Fh doses were found to be 200, 500, and 1000 mgFe/L for humic acid concentrates at UV254 of 1.5, 3.0, and 6.0 cm-1 respectively, whereas this value was 1000 mgFe/L for the seawater concentrates at UV254 of 1.5 cm-1. At these optimal doses, the recoveries of spiked AiV and Qß ranged from 2% to 12% in the humic acid and seawater concentrates, respectively. In addition, high levels of indigenous viruses (including AiV ≥ 2.81 log10 copies/mL and pepper mild mottle virus ≥ 2.77 log10 copies/mL) were detected from seawater concentrates after Fh treatment, while none were detected without Fh treatment. Fh treatment effectively mitigated the inhibitive effects from environmental water samples.