Evaluation of virus removal in membrane bioreactor (MBR) and conventional activated sludge (CAS) processes based on long-term monitoring at two wastewater treatment plants.

The membrane bioreactor (MBR) process always offers better wastewater treatment than conventional activated sludge (CAS) treatment. However, the difference in their efficacy of virus reduction remains unknown. To investigate this, we monitored virus concentrations before and after MBR and CAS processes over 2 years. Concentrations of norovirus genotypes I and II (NoV GI and GII), aichivirus (AiV), F-specific RNA phage genotypes I, II, and III (GI-, GII-, and GIII-FRNAPHs), and pepper mild mottle virus (PMMoV) were measured by a quantitative polymerase chain reaction (qPCR) method at two municipal wastewater treatment plants (WWTPs A and B) in Japan. Virus concentration datasets containing left-censored data were estimated by using both maximum likelihood estimation (MLE) and robust regression on order statistics (rROS) approaches. PMMoV was the most prevalent at both WWTPs, with median concentrations of 7.5 to 8.8 log10 copies/L before treatment. Log10 removal values (LRVs) of all viruses based on means and standard deviations of concentrations before and after treatment were consistently higher following MBR than following CAS. We used NoV GII as a model pathogen in a quantitative microbial risk assessment of the treated water, and we estimated the additional reductions required following MBR and CAS processes to meet the guideline of 10-6 DALYs pppy for safe wastewater reuse.

Comparison on removal performance of virus, antibiotic-resistant bacteria, cell-associated and cell-free antibiotic resistance genes, and indicator chemicals by ozone in the filtrated secondary effluent of a sewage treatment plant.

Due to the widespread appearance of viruses, antibiotic-resistant bacteria (ARBs), and antibiotic resistance genes (ARGs) in the aquatic environment, more powerful oxidation processes such as ozonation are needed to enhance the efficiency of their inactivation and removal during wastewater treatment. However, information is lacking on the elimination rates of viruses, ARBs, cell-associated ARGs (ca-ARGs), and cell-free ARGs (cf-ARGs) during ozonation. This study examined the kinetics and dose-dependent inactivation of a virus (MS2 coliphage) and an ARB (Ampicillin-resistant [AmpR] E. coli) and the removal of ca- and cf-ARGs (plasmid-encoded blaTEM) by ozonation in a filtered secondary effluent (SE) of a municipal sewage treatment plant (STP). In addition, the ozonation kinetics of carbamazepine (CBZ) and metoprolol (MTP)-ubiquitous organic micropollutants with different removal rate constants-were also investigated in order to monitor their effectiveness as indicators for the abovementioned biological risk factors. Our results showed that ozonation was an efficient way to remove MS2, AmpRE. coli, ARGs, CBZ, and MTP. We investigated the kinetics of their inactivation/removal with respect to exposure in terms of CT (dissolved ozone concentration C and contact time T) value, and found their inactivation/removal constants were in the following order: MS2 (8.66 ×103 M-1s-1) ≈ AmpRE. coli (8.19 ×103 M-1s-1) > cf-ARG (3.95 ×103 M-1s-1) > CBZ (3.21 ×103 M-1s-1) > ca-ARG (2.48×103 M-1s-1) > MTP (8.35 ×102 M-1s-1). In terms of specific ozone dose, > 5-log inactivation of MS2 was observed at > 0.30 mg O3/mg DOC, while > 5-log inactivation of AmpRE. coli was confirmed at 1.61-2.35 mg O3/mg DOC. Moreover, there was almost no removal of ca-ARG when the specific ozone dose was < 0.68 mg O3/mg DOC. However, 2.86-3.42-log removal of ca-ARG was observed at 1.27-1.31 mg O3/mg DOC, while 1.14-1.36-log removal of cf-ARG was confirmed at 3.60-4.30 mg O3/mg DOC. As alternative indicators, > 4-log removal of CBZ was observed at > 1.00 mg O3/mg DOC, while > 2-log removal of MTP was confirmed at > 2.00 mg O3/mg DOC. Thus, it was observed that inactivation of E. coli needs a greater ozone dose to achieve the same level of inactivation of AmpRE. coli; for ARGs, cf-ARG can persist longer than ca-ARG if low dosages of ozone are applied in the filtrated SE, CBZ might act as an indicator with which to monitor the inactivation of viruses and ARBs, while MTP might act as an indicator with which to monitor removal of ARGs. Moreover, cf-ARG cannot be neglected even after ozonation due to the possibility that ca-ARGs can become cf-ARGs during ozonation and be discharged with the final effluent, posing a potential risk to the receiving environment.

Antimicrobial resistome and mobilome in the urban river affected by combined sewer overflows and wastewater treatment effluent.

The dissemination of antimicrobial resistance in the environment is an emerging global health problem. Wastewater treatment effluent and combined sewer overflows (CSOs) are major sources of antimicrobial resistance in urban rivers. This study aimed to clarify the effect of municipal wastewater treatment effluent and CSO on antimicrobial resistance genes (ARGs), mobile gene elements, and the microbial community in an urban river. The ARG abundance per 16S-based microbial population in the target river was 0.37-0.54 and 0.030-0.097 during the CSO event and dry weather, respectively. During the CSO event, the antimicrobial resistome in the river shifted toward a higher abundance of ARGs to clinically important drug classes, including macrolide, fluoroquinolone, and ß-lactam, whereas ARGs to sulfonamide and multidrug by efflux pump were relatively abundant in dry weather. The abundance of intI1 and tnpA genes were highly associated with the total ARG abundance, suggesting their potential application as an indicator for estimating resistome contamination. Increase of prophage during the CSO event suggested that impact of CSO has a greater potential for horizontal gene transfer (HGT) via transduction. Consequently, CSO not only increases the abundance of ARGs to clinically important antimicrobials but also possibly enhances potential of HGT in urban rivers.

Tracking community infection dynamics of COVID-19 by monitoring SARS-CoV-2 RNA in wastewater, counting positive reactions by qPCR.

Wastewater-based epidemiology has proved useful for monitoring the COVID-19 infection dynamics in communities. However, in regions of low prevalence, low concentrations of SARS-CoV-2 RNA in wastewater make this difficult. Here, we used real-time reverse-transcription PCR (RT-qPCR) to monitor SARS-CoV-2 RNA in wastewater from October 2020 to December 2022 during the third, fourth, fifth, sixth, seventh, and eighth waves of the COVID-19 outbreak in Japan. Viral RNA was below the limit of detection in all samples during the third and fourth waves. However, by counting the number of positive replicates in qPCR of each sample, we found that the positive ratio to all replicates in wastewater was significantly correlated with the number of clinically confirmed cases by the date of symptom onset during the third, fourth, and fifth waves. Time-step analysis indicated that, for 2 days either side of symptom onset, COVID-19 patients excreted in their feces large amounts of virus that wastewater surveillance could detect. We also demonstrated that the viral genome copy number in wastewater, as estimated from the positive ratio of SARSA-CoV-2 RNA, was correlated with the number of clinically confirmed cases. The positive count method is thus useful for tracing COVID-19 dynamics in regions of low prevalence.

Reduction of SARS-CoV-2 by biological nutrient removal and disinfection processes in full-scale wastewater treatment plants.

Detection of SARS-CoV-2 RNA in wastewater poses people's concerns regarding the potential risk in water bodies receiving wastewater treatment effluent, despite the infectious risk of SARS-CoV-2 in wastewater being speculated to be low. Unlike well-studied nonenveloped viruses, SARS-CoV-2 in wastewater is present abundantly in both solid and liquid fractions of wastewater. Reduction of SARS-CoV-2 in past studies were likely underestimated, as SARS-CoV-2 in influent wastewater were quantified in either solid or liquid fraction only. The objectives of this study were (i) to clarify the reduction in SARS-CoV-2 RNA during biological nutrient removal and disinfection processes in full-scale WWTPs, considering the SARS-CoV-2 present in both solid and liquid fractions of wastewater, and (ii) to evaluate applicability of pepper mild mottle virus (PMMoV) as a performance indicator for reduction of SARS-CoV-2 in WWTPs. Accordingly, large amount of SARS-CoV-2 RNA were partitioned in the solid fraction of influent wastewater for composite sampling than grab sampling. When SARS-CoV-2 RNA in the both solid and liquid fractions were considered, log reduction values (LRVs) of SARS-CoV-2 during step-feed multistage biological nitrogen removal (SM-BNR) and enhanced biological phosphorus removal (EBPR) processes ranged between>2.1-4.4 log and did not differ significantly from those in conventional activated sludge (CAS). The LRVs of SARS-CoV-2 RNA in disinfection processes by ozonation and chlorination did not differ significantly. PMMoV is a promising performance indicator to secure reduction of SARS-CoV-2 in WWTPs, because of its higher persistence in wastewater treatment processes and abundance at a detectable concentration even in the final effluent after disinfection.

Eyestalk transcriptome and methyl farnesoate titers provide insight into the physiological changes in the male snow crab, Chionoecetes opilio, after its terminal molt.

The snow crab, Chionoecetes opilio, is a giant deep-sea brachyuran. While several decapod crustaceans generally continue to molt and grow throughout their lifetime, the snow crab has a fixed number of molts. Adolescent males continue to molt proportionately to their previous size until the terminal molt at which time an allometric increase in chela size occurs and an alteration of behavioral activities occurs, ensuring breeding success. In this study, we investigated the circulating concentrations of methyl farnesoate (an innate juvenile hormone in decapods) (MF) before or after the terminal molt in males. We then conducted eyestalk RNAseq to obtain molecular insight into the regulation of physiological changes after the terminal molt. Our analyses revealed an increase in MF titers after the terminal molt. This MF surge may be caused by suppression of the genes that encode MF-degrading enzymes and mandibular organ-inhibiting hormone that negatively regulates MF biosynthesis. Moreover, our data suggests that behavioral changes after the terminal molt may be driven by the activation of biogenic amine-related pathways. These results are important not only for elucidating the physiological functions of MFs in decapod crustaceans, which are still largely unknown, but also for understanding the reproductive biology of the snow crab.

Biological-Activity-Based Prioritization of Antidepressants in Wastewater in England and Japan.

Antidepressants are one of the most commonly prescribed pharmaceuticals. Although they have been frequently detected in aquatic environments around the globe, little is known regarding their adverse effects on humans and aquatic organisms. Recently, an in vitro monoamine transporter inhibition assay was developed to detect transporter-inhibitory activities of antidepressants in wastewater in Japan. However, it was unclear which antidepressants were responsible for transporter-inhibitory activities in wastewater. Herein, the per capita consumption of 32 antidepressants, their excretion of unchanged parent compounds, per capita water consumption, removal rate during wastewater treatment processes, and potency values from the monoamine transporter inhibition assay were used to prioritize antidepressants of concern in effluent wastewater in England and Japan. In both countries, sertraline and O-desmethylvenlafaxine had the highest contribution to inhibitory activities against the human serotonin transporter (hSERT) and zebrafish SERT (zSERT), respectively. It was found that the antidepressants inhibited the zSERT more strongly than the hSERT. The inhibitory activities found against the zSERT in wastewater in England and Japan were higher than thresholds for abnormal behavior in fish. The antidepressants prioritized in this study provide insight into launching environmental monitoring and ecotoxicological studies of antidepressants.

Development of two microbial source tracking markers for detection of wastewater-associated Escherichia coli isolates.

Escherichia coli has been used as an indicator of fecal pollution in environmental waters. However, its presence in environmental waters does not provide information on the source of water pollution. Identifying the source of water pollution is paramount to be able to effectively reduce contamination. The present study aimed to identify E. coli microbial source tracking (MST) markers that can be used to identify domestic wastewater contamination in environmental waters. We first analyzed wastewater E. coli genomes sequenced by us (n = 50) and RefSeq animal E. coli genomes of fecal origin (n = 82), and identified 144 candidate wastewater-associated marker genes. The sensitivity and specificity of the candidate marker genes were then assessed by screening the genes in 335 RefSeq wastewater E. coli genomes and 3318 RefSeq animal E. coli genomes. We finally identified two MST markers, namely W_nqrC and W_clsA_2, which could be used for detection of wastewater-associated E. coli isolates. These two markers showed higher performance than the previously developed human wastewater-associated E. coli markers H8 and H12. When used in combination, W_nqrC and W_clsA_2 showed specificity of 98.9 % and sensitivity of 25.7 %. PCR assays to detect W_nqrC and W_clsA_2 were also developed and validated. The developed PCR assays are potentially useful for detecting E. coli isolates of wastewater origin in environmental waters, though users should keep in mind that the sensitivity of these markers is not high. Further studies are needed to assess the applicability of the developed markers to a culture-independent approach.

Removal performance of SARS-CoV-2 in wastewater treatment by membrane bioreactor, anaerobic-anoxic-oxic, and conventional activated sludge processes.

The potential risk of SARS-CoV-2 in treated effluent from a wastewater treatment plant (WWTP) is concerned since SARS-CoV-2 is contained in wastewater during the COVID-19 outbreak. However, the removal of SARS-CoV-2 in WWTP has not been well investigated. The objectives of this study were (i) to clarify the removal performance of SARS-CoV-2 during wastewater treatment, (ii) to compare the removal performance of different secondary treatment processes, and (iii) to evaluate applicability of pepper mild mottle of virus (PMMoV) as a performance indicator for the reduction of SARS-CoV-2 RNA in wastewater treatment. Influent wastewater, secondary-treatment effluent (before chlorination), and final effluent (after chlorination) samples were collected from a WWTP from May 28 to September 24, 2020, during the COVID-19 outbreak in Japan. The target WWTP had three parallel treatment systems employing conventional activated sludge (CAS), anaerobic-anoxic -oxic (A2O), and membrane bioreactor (MBR) processes. SARS-CoV-2 in both the liquid and solid fractions of the influent wastewater was concentrated and quantified using RT-qPCR. SARS-CoV-2 in treated effluent was concentrated from 10 L samples to achieve a detection limit as low as 10 copies/L. The log reduction value (LRV) of SARS-CoV-2 was 2.7 ± 0.86 log10 in CAS, 1.6 ± 0.50 log10 in A2O, and 3.6 ± 0.62 log10 in MBR. The lowest LRV observed during the sampling period was 2.8 log10 in MBR, 1.2 log10 in CAS, and 1.0 log10 in A2O process, indicating that the MBR had the most stable reduction performance. PMMoV was found to be a good indicator virus to evaluate reduction performance of SARS-CoV-2 independent of the process configuration because the LRV of PMMoV was significantly lower than that of SARS-CoV-2 in the CAS, A2O and MBR processes.

Occurrence of E. coli and antibiotic-resistant E. coli in the southern watershed of Lake Biwa, including in wastewater treatment plant effluent and inflow rivers.

The emergence of antibiotic-resistant bacteria (ARB) and their antibiotic resistance genes (ARGs) poses a serious challenge to human, animal, and environmental health worldwide. ARB can spread into the environment via various sources and routes. In this study, we investigated the occurrence of antibiotic-resistant E. coli in the southern watershed of Lake Biwa. Two-year monitoring of antibiotic-resistant E. coli was carried out in the southern part of Lake Biwa and inflow rivers and at three WWTPs around the southern part of the lake. Concentrations of E. coli in waters that are resistant to ampicillin (AMP), cefotaxime (CTX), ceftazidime (CAZ), levofloxacin (LVFX), tetracycline (TC), and amikacin (AMK) were measured using the culture method. Of these antibiotic-resistant E. coli, AMP-resistant E. coli were found at the highest prevalence, followed by LVFX, CTX, CAZ, TC, and AMK-resistant in both the influent and effluent of WWTPs. These resistance patterns in wastewater are the same as those in clinical samples in Japan. The numbers of antibiotic-resistant E. coli decreased by around a factor of 1000 during the wastewater treatment processes, but the rates clearly increased, suggesting that selection for antibiotic resistance might occur during the wastewater treatment process. AMP-resistant and TC-resistant E. coli were also detected in Lake Biwa and inflow rivers, which suggests that antibiotic resistance might come from not only WWTPs but also livestock farms and small-scale wastewater treatment facilities located in the river catchment.

Emergence of rare carbapenemases (FRI, GES-5, IMI, SFC and SFH-1) in Enterobacterales isolated from surface waters in Japan.

OBJECTIVES: Carbapenemase-producing Enterobacterales (CPE) pose serious threats to public health. Compared with clinical CPE, the genetic characteristics of environmental CPE are not well understood. This study aimed to characterize the genetic determinants of carbapenem resistance in CPE isolated from environmental waters in Japan. METHODS: Eighty-five water samples were collected from rivers and a lake in Japan. CPE were identified using selective media, and genome sequencing was performed for the obtained isolates (n = 21). RESULTS: Various rare/novel carbapenemases were identified: GES-5 in Raoultella planticola (n = 1), FRI-8 and FRI-11 in Enterobacter spp. (n = 8), IMI-22 and IMI-23 in Serratia ureilytica (n = 3), and SFC-1, SFC-2 and SFH-1 in Serratia fonticola (n = 9). Genomes of 11 isolates could be closed, allowing the elucidation of the genetic contexts of the carbapenemase genes. The blaGES-5 gene was located within a class 1 integron, In2071 (cassette array, blaGES-5-aacA3-aadA16), on a 33 kb IncP6 plasmid. The blaFRI-8 genes were carried on IncFII(Yp) plasmids ranging in size from 191 kb to 244 kb, and the blaFRI-11 genes were carried on 70 kb and 74 kb IncFII(pECLA)/IncR plasmids. The blaIMI-22 and blaIMI-23 genes were co-located on a 107 kb plasmid. The blaSFC and blaSFH-1 genes were found on putative genomic islands inserted at tRNA-Phe genes in chromosomes. CONCLUSIONS: This study revealed the presence of rare/novel carbapenemases among CPE in aquatic environments, suggesting that the environment may act as a potential reservoir of these minor carbapenemases.

Characterization of nitrosamines and nitrosamine precursors as non-point source pollutants during heavy rainfall events in an urban water environment.

N-nitrosodimethylamine (NDMA) and N-nitrosomorpholine (NMOR) and their specific precursors (N,N-dimethylformamide [DMF] for NDMA and morpholine [MOR] for NMOR) were widely identified in runoff of urban area around the southern Lake Biwa basin, Japan. It was thought that this runoff might constitute a non-point source of the four compounds in rivers and sewage treatment plants (STPs) during heavy rainfall events. We investigated the spatiotemporal patterns of NDMA, NMOR, DMF and MOR in runoff and rivers in rainy days. NDMA and NMOR were detected in concentrations of up to 295 ng/L, while DMF and MOR were detected in concentrations of up to 33.7 µg/L. Continuous sequential sampling over periods of 24 or 48 h at the largest STP in the study area revealed that the four compounds in the primary effluent (PE) each had higher mass fluxes during heavy rainfall events than on dry days. This phenomenon might be contributed to non-point sources (e.g., runoff) from infiltration/inflow related to rainwater into sanitary sewers. Moreover, the four compounds were confirmed to have higher mass fluxes in the final effluent of the STP during periods of PE bypass (1.3-1.7 times for NDMA, NMOR, and MOR; over 200 times for DMF; on average) than that on dry days because of increasing inflow during heavy rain than during periods without PE bypass in dry weather.

Comparison of five polyethylene glycol precipitation procedures for the RT-qPCR based recovery of murine hepatitis virus, bacteriophage phi6, and pepper mild mottle virus as a surrogate for SARS-CoV-2 from wastewater.

Polyethylene glycol (PEG) precipitation is one of the conventional methods for virus concentration. This technique has been used to detect severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA in wastewater. The procedures and seeded surrogate viruses were different among implementers; thus, the reported whole process recovery efficiencies considerably varied among studies. The present study compared five PEG precipitation procedures, with different operational parameters, for the RT-qPCR-based whole process recovery efficiency of murine hepatitis virus (MHV), bacteriophage phi6, and pepper mild mottle virus (PMMoV), and molecular process recovery efficiency of murine norovirus using 34 raw wastewater samples collected in Japan. The five procedures yielded significantly different whole process recovery efficiency of MHV (0.070%-2.6%) and phi6 (0.071%-0.51%). The observed concentration of indigenous PMMoV ranged from 8.9 to 9.7 log (8.2 × 108 to 5.6 × 109) copies/L. Interestingly, PEG precipitation with 2-h incubation outperformed that with overnight incubation partially due to the difference in molecular process recovery efficiency. The recovery load of MHV exhibited a positive correlation (r = 0.70) with that of PMMoV, suggesting that PMMoV is the potential indicator of the recovery efficiency of SARS-CoV-2. In addition, we reviewed 13 published studies and found considerable variability between different studies in the whole process recovery efficiency of enveloped viruses by PEG precipitation. This was due to the differences in operational parameters and surrogate viruses as well as the differences in wastewater quality and bias in the measurement of the seeded load of surrogate viruses, resulting from the use of different analytes and RNA extraction methods. Overall, the operational parameters (e.g., incubation time and pretreatment) should be optimized for PEG precipitation. Co-quantification of PMMoV may allow for the normalization of SARS-CoV-2 RNA concentration by correcting for the differences in whole process recovery efficiency and fecal load among samples.

Spatial and temporal distributions of enteric viruses and indicators in a lake receiving municipal wastewater treatment plant discharge.

Although lake water can be used as a source of drinking water and recreational activities, there is a dearth of research on the occurrence and fate of enteric viruses. Over a period of 14 months at six points in 2014-2015, we conducted monthly monitoring of the virological water quality of a Japanese lake. The lake receives effluent from three surrounding wastewater treatment plants and retains water for about two weeks. These features allowed us to investigate the occurrence and fate of viruses in the lake environment. Human enteric viruses such as noroviruses and their indicators (pepper mild mottle virus and F-specific RNA bacteriophage [FRNAPH] genogroups) were quantified by PCR-based assays. Additionally, FRNAPH genogroups were quantified by infectivity-based assays to estimate the degree of virus inactivation. Pepper mild mottle virus, genogroup II (GII) norovirus, and GI-FRNAPH were identified in relatively high frequencies (positive in >40% out of 64 samples), with concentrations ranging from 1.3 × 101 to 2.9 × 104 copies/L. Human enteric viruses and some indicators were not detected and less prevalent, respectively, after April 2015. Principal component analysis revealed that the virological water quality changed gradually over time, but its differences between the sampling points were not apparent. FRNAPH genogroups were inactivated during the warm season (averaged water temperature of >20 °C) compared to the cool season (averaged water temperature of <20 °C), which may have been due to the more severe environmental stresses such as sunlight and water temperature. This suggests that the infection risk associated with the use of the lake water may have been overestimated by the gene quantification assay during the warm season.

Proposal for fluorescence-based in vitro assay using human and zebrafish monoamine transporters to detect biological activities of antidepressants in wastewater.

Antidepressants are among the most commonly detected pharmaceuticals in the aquatic environment. As they modulate neurotransmission in nervous systems, behavioural abnormalities among aquatic species are of concern. It is possible to measure the concentrations of selected antidepressants by chemical analysis, but other non-target antidepressants and active metabolites might also be present. Here, we propose an "in vitro monoamine transporter inhibition assay" to measure the biological activity of antidepressants, particularly monoamine transporter inhibitors, in wastewater. We used APP, a fluorescent substrate for monoamine transporters, to measure the activity of wastewater extracts at inhibiting APP uptake through the human serotonin transporter (hSERT), norepinephrine transporter (hNET), and dopamine transporter, and the zebrafish SERT (zSERT). We confirmed that the assay could measure the biological activity of test antidepressants. Interestingly, the IC50 values of antidepressants (the concentration that gave a 50% reduction of APP uptake) for the zSERT were smaller than those for the hSERT. For example, IC50 value of desipramine for the zSERT was 1/200 of that for the hSERT. These results indicate that antidepressants inhibited zSERT more strongly than hSERT. Then we applied the assay to extracts of effluent from municipal wastewater treatment plants and detected biological activity of antidepressants specifically against the hSERT, hNET, and zSERT for the first time. For the hSERT, antidepressant-equivalent quantities (EQs) ranged from 2.2 × 101 to 2.5 × 102 ng-clomipramine-EQ/L. For the hNET, EQs ranged from below limit of detection to 8.2 × 101 ng-desipramine-EQ/L. For the zSERT, EQs ranged from 2.8 × 102 to 3.3 × 102 ng-duloxetine-EQ/L. The in vitro monoamine transporter inhibition assay is thus useful for measuring the biological activity of antidepressants in the aquatic environment.

Characterization of G protein-coupled estrogen receptors in Japanese medaka, Oryzias latipes.

The G protein-coupled estrogen receptor 1 (Gper1) is a membrane-bound estrogen receptor that mediates non-genomic action of estrogens. A Gper1-mediating pathway has been implicated in reproductive activities in fish, including oocyte growth, but Gper1 has been characterized in only a very limited number of fish species. In this study, we cloned and characterized two genes encoding medaka (Oryzias latipes) Gper1s, namely, Gper1a and Gper1b, and phylogenic and synteny analyses suggest that these genes originate through a teleost-specific whole genome duplication event. We found that Gper1a induced phosphorylation of mitogen-activated protein kinase (MAPK) in 293T cells transfected with medaka Gper1s on exposure to the natural estrogen, 17ß-estradiol (E2) and a synthetic Gper1 agonist (G-1), and treatment with both E2 and G-1 also decreased the rate of spontaneous maturation in medaka oocytes. These findings show that the processes for oocyte growth and maturation are sensitive to estrogens and are possibly mediated through Gper1a in medaka. We also show that 17α-ethinylestradiol (EE2), one of the most potent estrogenic endocrine-disrupting chemicals, and bisphenol A (BPA, a weak environmental estrogen) augmented phosphorylation of MAPK through medaka Gper1s in 293T cells. Interestingly, however, treatment with EE2 or BPA did not attenuate maturation of medaka oocytes. Our findings support that Gper1-mediated effects on oocytes are conserved among fish species, but effects of estrogenic endocrine-disrupting chemicals on oocytes acting through Gper1 may be divergent among fish species.

Wastewater-derived antagonistic activities of G protein-coupled receptor-acting pharmaceuticals in river water.

Pharmaceuticals are widely detected in aquatic environments, and their potential risks to aquatic species are of concern because they are designed to be biologically active. Here, we used an in vitro assay, called the transforming growth factor α shedding assay, to measure the biological activities of G protein-coupled receptor (GPCR)-acting pharmaceuticals present in river water and effluents from municipal wastewater treatment plants (WWTPs) in Japan from 2014 to 2016. Antagonistic activities against angiotensin (AT1), dopamine (D2), adrenergic (ß1), acetylcholine (M1) and histamine (H1) receptors were detected in river water, and were stronger downstream than upstream owing to effluent from WWTPs along the river. Ozonation at one WWTP reduced these activities. Concentrations of sulpiride (D2 antagonist) could explain 73% of antagonistic activities against the D2 receptor; those of metoprolol, atenolol and propranolol (ß1 antagonists) could explain 16% of activities against the ß1 receptor; and those of pirenzepine (M1 antagonist) could explain 15% of activities against the M1 receptor. Therefore, other receptor antagonists also occur. GPCR-acting pharmaceuticals should be given more attention in environmental monitoring and toxicity testing.

Biological Activity-Based Prioritization of Pharmaceuticals in Wastewater for Environmental Monitoring: G Protein-Coupled Receptor Inhibitors.

Pharmaceuticals raise concerns for aquatic species owing to their biological activities. It is estimated that nearly 40% of marketed pharmaceuticals target G protein-coupled receptors (GPCRs). Using an in vitro transforming growth factor-α (TGFα) shedding assay, we previously detected antagonistic activities of GPCR-acting pharmaceuticals against angiotensin (AT1), dopamine (D2), acetylcholine (M1), adrenergic family members (ß1), and histamine (H1) receptors at up to µg-antagonist-equivalent quantities/L in wastewater in England and Japan. However, which pharmaceuticals were responsible for biological activities in wastewater remained unclear. Here, we used (1) the consumption of GPCR-acting pharmaceuticals, particularly antagonists, as calculated from prescriptions, (2) their urinary excretion, and (3) their potency measured by the TGFα shedding assay to prioritize them for analysis in wastewater in England and Japan. We calculated predicted activities of 48 GPCR-acting pharmaceuticals in influents in England and Japan and identified which were mainly responsible for antagonistic activities in wastewater against each GPCR. Mixtures of pharmaceuticals tested in this study were confirmed to behave additively. The combination of consumption and potency is useful in prioritizing pharmaceuticals for environmental monitoring and toxicity testing.

Quantification of Pharmaceutical Related Biological Activity in Effluents from Wastewater Treatment Plants in UK and Japan.

While pharmaceuticals are now routinely detected in aquatic environments, we know little of the biological activity their presence might provoke. It is estimated that nearly 40% of all marketed pharmaceuticals are G protein-coupled receptors (GPCRs) acting pharmaceuticals. Here, we applied an in-vitro assay, called the TGFα shedding assay, to measure the biological activities of GPCRs-acting pharmaceuticals present in effluents from municipal wastewater treatment plants in the United Kingdom (UK) and Japan from 2014 to 2016. The results indicated that compounds were present in the wastewater with antagonistic activities against angiotensin (AT1), dopamine (D2), adrenergic (ß1), acetylcholine (M1), and histamine (H1) receptors in both countries. The most consistent and powerful antagonistic activity was against the H1, D2, and AT1 receptors at up to microgram-antagonist-equivalent quantity/L. Chemical analysis of the same UK samples was also conducted in parallel. Comparing the results of the bioassay with the chemical analysis indicated (1) the existence of other D2 or M1 receptor antagonists besides sulpiride (D2 antagonist) or pirenzepine (M1 antagonist) in wastewater and (2) that there might be a mixture effect between agonist and antagonistic activities against ß1 receptor. GPCR-acting pharmaceuticals should be paid more attention in the environmental monitoring and toxicity testing in future studies.

Comprehensive Study on Enteric Viruses and Indicators in Surface Water in Kyoto, Japan, During 2014-2015 Season.

Certain enteric viruses that are present in the water environment are potential risk factors of waterborne infections. To better understand the impact of viruses in water, both enteric viruses and their potential indicators should be comparatively investigated. In this study, occurrences of GI- and GII-noroviruses (NoVs), sapovirus (SaV), rotavirus (RoV), Aichi virus 1 (AiV-1), enterovirus (EV), and pepper mild mottle virus (PMMoV) were quantitatively determined in surface water samples in Japan. Additionally, the genotype distribution of GI- and GII-NoVs was determined using a next-generation amplicon sequencing. PMMoV was the most abundant virus regardless of season and location, indicating its usefulness as an indicator for the viral contamination of water. Other potential indicators, AiV and EV, were less abundant than GII-NoV. Viruses other than PMMoV showed seasonality, i.e., EV and other viruses (NoVs, SaV, RoV, and AiV-1) became prevalent during summer and winter, respectively. SaV showed a relatively high abundance at a location that was affected by untreated wastewater. Regarding NoV genotypes, GI.1, GI.2, GI.4, GI.5, GI.6, GII.3, GII.4, GII.6, and GII.17 were found from the surface water samples. GII.4 and GII.17 seemed to have contributed to the high abundance of GII-NoV in the samples. Interestingly, GII.17 strains became prevalent in the water samples before becoming prevalent among gastroenteritis patients in Japan. These findings provide further insights into the properties of viruses as contaminants in the water environment.