Localized pollution of veterinary antibiotics in watersheds receiving treated effluents from swine farms.

Swine excrement is discharged into surface waters mainly as effluent in Asian countries. As swine production consumes more antibiotics and less water than humans, a mismatch of the size of swine farms and that of the rivers receiving their effluent could create severe pollution by antibiotics. However, little is known about the occurrence of antibiotics in such rivers. We therefore monitored seven veterinary drugs, six human drugs (including a metabolite), three drugs for both use (including a metabolite), and major water qualities at 30 sites in Japanese watersheds where swine outnumber humans and where their excrement is largely treated on-site by aerobic biological wastewater processes. The compositions of veterinary drugs differed substantially among sites, unlike human drugs, indicating various patterns of use among swine farms. Median concentrations at the 30 sites were <1 ng/L for seven out of the ten drugs used in livestock, whereas maximum concentrations were >1000 ng/L for three and 100-1000 ng/L for four of them, giving median-maximum among the sites of >3 log for two and 2-3 log for six of them. The spatial distribution ranges of concentrations of veterinary drugs were wider than those of human drugs (mostly <1.5 log) and other analytes (mostly <1 log), despite the correlation between those of total veterinary drugs and nitrogen, attributable to fewer swine farms than households, the intensive animal husbandry, and the various drug-use patterns among the farms. The range of maximum concentrations of veterinary drugs in the watersheds was comparable to those reported in other Asian watersheds with less strict management of swine excrement, attributable to their slow decay in conventional wastewater treatment on swine farms. Thus, attention should be paid to hot-spot pollution of antibiotics on large Asian swine farms adjacent to streams with limited dilution capacity.

Applicability of F-specific bacteriophage subgroups, PMMoV and crAssphage as indicators of source specific fecal contamination and viral inactivation in rivers in Japan.

To date, several microbes have been proposed as potential source-specific indicators of fecal pollution. 16S ribosomal RNA gene markers of the Bacteroidales species are the most widely applied due to their predominance in the water environment and source specificity. F-specific bacteriophage (FPH) subgroups, especially FRNA phage genogroups, are also known as potential source-specific viral indicators. Since they can be quantified by both culture-based and molecular assays, they may also be useful as indicators for estimating viral inactivation in the environment. Pepper mild mottle virus (PMMoV) and crAssphage, which are frequently present in human feces, are also potentially useful as human-specific indicators of viral pollution. This study aimed to evaluate the applicability of FPH subgroups, PMMoV, and crAssphage as indicators of source-specific fecal contamination and viral inactivation using 108 surface water samples collected at five sites affected by municipal and pig farm wastewater. The host specificity of the FPH subgroups, PMMoV, and crAssphage was evaluated by principal component analysis (PCA) along with other microbial indicators, such as 16S ribosomal RNA gene markers of the Bacteroidales species. The viabilities (infectivity indices) of FRNA phage genogroups were estimated by comparing their numbers determined by infectivity-based and molecular assays. The PCA explained 58.2% of the total information and classified microbes into three groups: those considered to be associated with pig and human fecal contamination and others. Infective and gene of genogroup IV (GIV)-FRNA phage were assumed to be specific to pig fecal contamination, while the genes of GII-FRNA phage and crAssphage were identified to be specific to human fecal contamination. However, PMMoV, infective GI-FRNA phage, and FDNA phage were suggested to not be specific to human or pig fecal contamination. FRNA phage genogroups, especially the GIV-FRNA phage, were highly inactivated in the warm months in Japan (i.e., July to November). Comparing the infectivity index of several FRNA phage genogroups or other viruses may provide further insight into viral inactivation in the natural environment and by water treatments.

Spatiotemporal distribution of veterinary and human drugs and its predictability in Japanese catchments.

Little is known about the predictability of mass flows of veterinary drugs in Asian catchments, where effluent from livestock farms is a major source. We therefore conducted this study to understand the applicability and limitations of a population-based emission model, which assumed usage of veterinary and human drugs to be evenly distributed over the national livestock or human population throughout the year, and sources to be effluent discharges at livestock farms, households, and sewage treatment plants in Japanese catchments. We monitored five veterinary drugs (lincomycin, sulfamonomethoxine, tiamulin, tylosin, and tilmicosin), two human and livestock drugs (sulfamethoxazole and trimethoprim), two human drugs (carbamazepine and clarithromycin), and a metabolite (sulfapyridine) of a human drug once a month over 2 years in eight Japanese rivers which have active livestock farming in their catchments. Mass flows of carbamazepine and sulfapyridine were stable, while those of veterinary drugs fluctuated widely, especially sulfamonomethoxine and tilmicosin, whose 25 %-100 % ranges averaged 1.5 and 1.2 log units, respectively, attributable mainly to their usage patterns. The model accurately predicted mean mass flows of carbamazepine in the rivers with errors of <±0.3 log unit. Although it slightly to moderately overestimated those of the other four human-related compounds, the incorporation of an empirical correction factor, determined to minimize mean absolute error (MAE) among the rivers, substantially lowered their MAEs to <0.23 log units. However, the MAEs of the five veterinary drugs were as high as 0.42 (sulfamonomethoxine) to 0.60 (tiamulin) log units even with the coefficient, likely due mainly to the spatial distribution of their usage per capita. So as not to overlook spatiotemporal elevation of risks of veterinary drugs, a stochastic method should be applied in their management. This is the first study to assess the use of spatiotemporal homogeneity in usage per capita of veterinary drugs in Asian catchments.

Complex behavior between microplastic and antibiotic and their effect on phosphorus-removing Shewanella strain during wastewater treatment.

Owing to their widespread application and use, microplastics (MPs) and antibiotics coexist in the sewage treatment systems. In this study, the effects and mechanisms of the combined stress of MPs and ciprofloxacin (CIP) on phosphorus removal by phosphorus-accumulating organisms (PAOs) were investigated. This study found that the four types of MPs and CIP exhibited different antagonistic effects on the inhibition of phosphorus removal by PAO. MPs reduced the effective concentration of CIP through adsorption and thus reduced its toxicity, which was affected by the biofilms on MPs. In addition, CIP may cause PAO to produce more extracellular polymeric substances, which reduces the physical and oxidative stress of MPs on PAO. Our results are helpful as they increase the understanding of the effects of complex emerging pollutants in sewage systems and propose measures to strengthen the biological phosphorus removal in sewage treatment processes.

In-stream sorption of azithromycin and levofloxacin in a river receiving sewage treatment plant effluent.

Modelling natural attenuation is crucial to managing pharmaceuticals. However, little is known about the mechanism behind their in-stream sorption. To better understand the in-stream attenuation of the highly sorptive antibiotics azithromycin (AZM) and levofloxacin (LVF), we monitored them in a 2.1-km stretch of the Asano River under diverse flow conditions. This stretch receives effluent directly from a sewage treatment plant (STP), which was a dominant source of the pharmaceuticals. Average distribution coefficients between dissolved and particulate phases (Kd,SPM) in the outflow river water were 6.3×105 L/kg for AZM and 7.5×104 L/kg for LVF, while those in the STP effluent were 1-2 orders of magnitude lower. Mass balances in the river stretch calculated by considering only dissolved phase (MBw) and both dissolved and particulate phases (MBs) were 8%-52% and 58%-102%, respectively, for AZM, and 58%-71% and 60%-105% for LVF. MBw

Toxicity and combined effects of antibiotics and nano ZnO on a phosphorus-removing Shewanella strain in wastewater treatment.

Antibiotics and nanoparticles, which are emerging contaminants, can occur simultaneously in biological wastewater treatment systems, potentially resulting in complex interactive effects. This study investigated the effects of individual and complex zinc oxide nanoparticles (nZnO) and antibiotics (quinolone and sulfonamide), on the Shewanella strain used to remove phosphorus (PO43-), metabolic processes, as well as its complexing and toxicity mechanisms. The inhibition of PO43- removal increased from 30.7% to 100.0% with increased nZnO concentrations (half maximal effective concentration (EC50) = 1.1 mg Zn/L) by affecting poly-p and glycogen metabolites. The combined exposure to nZnO and ciprofloxacin/norfloxacin (CIP/NOR) had a significant antagonistic effect on the removal of PO43- and on the metabolism of poly-p and glycogen in phosphate-accumulating organisms (PAOs), whereas the complexing of sulfonamide and nZnO had no significant additional effect. Thus, the complexing of nanoparticles and antibiotics exhibited different toxicity effects from the antibiotic structure-based complex reactions. These results can be used to improve wastewater treatment processes and reduce risks associated with wastewater discharge.

Modeling in-stream attenuation of N-nitrosodimethylamine and formaldehyde during urban river transportation based on seasonal and diurnal variation.

Disinfection by-products (DBPs) discharged from sewage treatment plants (STPs) could harm downstream receiving waters and drinking water resources. In-stream attenuation of photo- and non-photodegradable DBPs during river transportation is currently not well understood. Here we sought to fill this knowledge gap by meta-data-analysis for modeling in-stream attenuation of DBPs. Data were collected along a treated-wastewater-dominated 1.6-km stretch of a river channel for 3 years and incorporated seasonal and diurnal patterns. Photo-irradiation and water temperature were the main factors responsible for in-stream attenuation of photodegradable N-nitrosodimethylamine (NDMA), and water temperature for that of non-photodegradable formaldehyde (FAH). The factors were incorporated into photo-dependent and -independent models to account for temporal variations in NDMA and FAH, respectively. Estimated mass recoveries of NDMA and FAH agreed well with observed values along the stretch. The models developed here offer a novel and useful tool for estimating levels of NDMA and FAH during river transportation.

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.

Diurnal patterns of N-nitrosodimethylamine and formaldehyde behaviors in different seasons in surface water influenced by effluent from sewage treatment plants.

N-nitrosodimethylamine (NDMA) and formaldehyde (FAH) are probable carcinogenic disinfection by-products and have been found to occur in areas of the Yodo River basin that are influenced by treated wastewater. The diurnal patterns of them were examined with water samples collected over 4 years in different seasons at five outlets of four sewage treatment plants (STPs), seven main stream sites, and five tributary sites in the basin. Based on mass flux calculations, STPs were shown to be the main sources of the downstream for NDMA and FAH loads in the study area. Moreover, results revealed that photo degradable NDMA and non-photo degradable FAH showed different fates during the river transportation. For NDMA, in addition to direct photolysis, water temperature was identified as an important factor in NDMA attenuation in surface waters. NDMA attenuated significantly in the daytime and even during summer nights but persisted during winter nights, while attenuation of FAH was found to mainly be influenced by water temperature, and persisted during winter. Their behaviors were compared in an artificial river channel and whole study area, clearly demonstrating the importance of monitoring them in the water environment during periods of low UV intensity and water temperature, especially winter nights.

Predicting the sorption of azithromycin and levofloxacin to sediments from mineral and organic components.

Despite the strong association of azithromycin (AZM), a macrolide antibiotic, and levofloxacin (LVF), a quinolone antibiotic, to sediment, sorption data are scarce. We conducted sorption experiments with eight river sediments, their major clay minerals (illite and chlorite), a highly negatively charged clay mineral (montmorillonite), and an organic-matter-rich soil (Andosol). The sorption of AZM and LVF to the sediments was influenced by the concentration and type of coexisting inorganic cations as much as by reported organic cations. In addition, their linear sorption coefficients (Kd) to sediments were correlated with cation exchange capacity (CEC) but not organic carbon content, so cation exchange is the dominant sorption mechanism. Multiple linear regression analysis showed improved prediction of sediment Kd from CEC contributed by minerals and organic matter for AZM, but not for LVF. Kcec (= Kd/CEC) values of AZM were 2-3 orders of magnitude higher on minerals than on Andosol, but those of LVF ranged within a factor of 4. Therefore, mineral and organic components need to be separated in estimating AZM sorption to sediments. Sediment Kd values of AZM and LVF were satisfactorily predicted by a cation-exchange-based model using individual Kcec values on illite, chlorite, and Andosol (mean absolute error of 0.57 and 0.22 log units, respectively). Kcec values on montmorillonite and chlorite ranged within a factor of about 3 from those of illite for both antibiotics, and Kcec differences by mineral type would generally be negligible in model estimation. Because AZM was sorbed mostly to minerals in sediments, the model and sorption data can be applicable to various soils or sediments. Overall, the trend of LVF sorption corresponds to reported sorption of other organic cations, whereas remarkably higher AZM Kcec to minerals than to Andosol is attributable to its large lactone ring, higher molecular weight, or two charged amines.

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.

Source estimation of pharmaceuticals based on catchment population and in-stream attenuation in Yodo River watershed, Japan.

Fifty-five pharmaceuticals were monitored at four rivers and inlets and/or outlets of three sewage treatment plants (STPs) in Yodo River watershed, Japan over 17 sampling events. Twenty-six quantified pharmaceuticals were classified by source and fate. The load per person (LPP) of nine pharmaceuticals, including six with observed mass balance in studied river stretch of <80%, was appreciably lower in river water (RW) than in the effluent (EF) of STPs (RW/EF <0.5), indicating that they were susceptible to in-stream attenuation in the study area, while the others were relatively conservative. The LPP of 12 pharmaceuticals in RW were within ±50% of that in EF. Because their mass loadings in rivers were correlated with human population in the catchment and most people use the sewer system, the major source of the 12 pharmaceuticals was considered to be STPs. The LPP of the three most labile pharmaceuticals in STPs (caffeine, theophylline, and acetaminophen) was >1.5 in RW/EF and <1.0 in RW/influent (IF) of STPs. Poorly treated sewage discharged from households without using the sewer system was considered to be influential source of the three pharmaceuticals. The LPP (RW/EF) of caffeine, a pharmaceutical contained in food and beverage, was considerably higher than that of the other two, and this is attributable to untreated gray water discharged at households using the night-soil treatment system. The LPP of two veterinary drugs (sulfamonomethoxine and lincomycin) were >1.5 (RW/EF) and >1.0 (RW/IF). Their mass loadings in rivers showed a positive correlation with swine population in the catchment, although sulfamonomethoxine is equally used in both cattle and swine farming. This was attributable to application of cattle excrement as manure, and lability of sulfamonomethoxine during composting processes. The major source of the two veterinary drugs was considered to be on-site treatment facilities of swine urine.

[Detection of Physiological Activity of Pharmaceuticals in Wastewater and River Water].

　Pharmaceuticals are widely found in aquatic environments worldwide. Concern about their potential risks to aquatic species has been raised because they are designed to be biologically active. To address this concern, we must know whether biological activity of pharmaceuticals can be detected in waters. Nearly half of all marketed pharmaceuticals act by binding to the G protein-coupled receptor (GPCR). In this study, we measured the physiological activity of GPCR-acting pharmaceuticals in effluent from a wastewater treatment plant (WWTP) and upstream and downstream of its outfall in Japan during 2 years. We used the in vitro transforming growth factor-α (TGFα) shedding assay, which accurately and sensitively detects GPCR activation, to investigate the antagonistic activities of water extracts against receptors for dopamine (D2) and histamine (H1). Activities detected in waters were quantified as antagonist equivalent quantities (EQs). In WWTP effluent extracts, antagonistic activity was detected at several hundred ng/L of sulpiride-EQ (D2) and several µg/L of diphenhydramine (DIP)-EQ (H1). In downstream river water extracts, antagonistic activity against H1 was around several hundred ng/L of DIP-EQ, higher than that upstream owing to the WWTP effluent. This review discusses the research needed to resolve the concern about potential risks of pharmaceuticals in waters to aquatic species.

The different fate of antibiotics in the Thames River, UK, and the Katsura River, Japan.

Little is known about the mechanisms influencing the differences in attenuation of antibiotics between rivers. In this study, the natural attenuation of four antibiotics (azithromycin, clarithromycin, sulfapyridine, and sulfamethoxazole) during transport along the Thames River, UK, over a distance of 8.3 km, and the Katsura River, Japan, over a distance of 7.6 km was compared. To assist interpretation of the field data, the individual degradation and sorption characteristics of the antibiotics were estimated by laboratory experiments using surface water or sediment taken from the same rivers. Azithromycin, clarithromycin, and sulfapyridine were attenuated by 92, 48, and 11% in the Thames River stretch. The first-order decay constants of azithromycin and sulfapyridine were similar to those in the Katsura River, while that of clarithromycin was 4.4 times higher. For sulfamethoxazole, the attenuation was limited in both rivers. Loss of sulfapyridine was attributed to both direct and indirect photolysis in the Thames River, but to only direct photolysis in the Katsura River. Loss of azithromycin and clarithromycin was attributed to sorption to sediment in both rivers. The probable explanation behind the difference in loss rates of clarithromycin between the two rivers was considered to be sediment sorption capacity.

Linking changes in antibiotic effluent concentrations to flow, removal and consumption in four different UK sewage treatment plants over four years.

The arrival and discharge of seven antibiotics were monitored at two trickling filter sewage treatment plants of 6000 and 11,000 population equivalents (PE) and two activated sludge plants of 33,000 and 162,000 PE in Southern England. The investigation consisted of 24 h composite samples taken on two separate days every summer from 2012 to 2015 and in the winter of 2015 (January) from influent and effluent. The average influent concentrations generally matched predictions based on England-wide prescription data for trimethoprim, sulfamethoxazole, azithromycin, oxytetracycline and levofloxacin (within 3-fold), but were 3-10 times less for clarithromycin, whilst tetracycline influent concentrations were 5-17 times greater than expected. Over the four years, effluent concentrations at a single sewage plant varied by up to 16-fold for clarithromycin, 10-fold for levofloxacin and sulfamethoxazole, 7-fold for oxytetracycline, 6-fold for tetracycline, 4-fold for azithromycin and 3-fold for trimethoprim. The study attempted to identify the principal reasons for this variation in effluent concentration. By measuring carbamazepine and using it as a conservative indicator of transport through the treatment process, it was found that flow and hence concentration could alter by up to 5-fold. Measuring influent and effluent concentrations allowed assessments to be made of removal efficiency. In the two activated sludge plants, antibiotic removal rates were similar for the tested antibiotics but could vary by several-fold at the trickling filter plants. However, for clarithromycin and levofloxacin the variations in effluent concentration were above that which could be explained by either flow and/or removal alone so here year on year changes in consumption are likely to have played a role.

Assessing the population equivalent and performance of wastewater treatment through the ratios of pharmaceuticals and personal care products present in a river basin: Application to the River Thames basin, UK.

The quality of surface waters in lowland rivers is largely dependent on the efficiency of wastewater treatment. Even in the developed countries, there have been difficulties in evaluating the effectiveness of wastewater management and the proportion of wastewater content (WWC) in the river, as well as in estimating the contributing human population. This study aimed to develop a wastewater quality and quantity assessment based on the occurrence of pharmaceuticals in the receiving waters. A survey of 53 pharmaceuticals in 324 samples (river water and influent and effluent of sewage (wastewater) treatment plants) was carried out in southern England in the River Thames catchment over four years. Carbamazepine was selected as stable marker and from its concentration WWC in the rivers and cumulative human populations along the catchment were estimated. The estimated population had a strong relationship (R2=0.94) with that reported by the local water company. The concentration ratio of the labile marker caffeine to carbamazepine indicated the efficiency of wastewater treatment in the different treatment systems (i.e. trickling filter or activated sludge) and in the receiving waters. The ratio in some river samples revealed unexpected discharges of untreated or poorly treated wastewater, with a total concentration of the analytes (up to 20µg/L) five times higher than that in treated wastewater. Such information could be valuable to estimate the discharge or occurrence of not only non-targeted chemicals, but also pathogens within the basin.

Modeling the fate of a photoproduct of ketoprofen in urban rivers receiving wastewater treatment plant effluent.

Photoproducts of pharmaceuticals have been studied in order not to overlook their potential risks to aquatic organisms. However, no studies have verified an equation for predicting the fate of photoproducts in aquatic environment (Poiger equation) by field measurements, leaving uncertainties in its practical utility. Therefore, we conducted this study to test the applicability of the Poiger equation to 3-ethylbenzophenone (EBP), a photoproduct of ketoprofen (KTP). Photolysis experiments determined the fraction of KTP transformed into EBP as 0.744±0.074 and the quantum yield of EBP degradation as 0.000418±0.000090. Field studies in urban rivers and wastewater treatment plants (WWTPs) revealed that EBP was produced by sunlight, mainly in the rivers, but also appreciably in outdoor primary and secondary clarifiers in the WWTPs. We developed a model in the secondary clarifiers, disinfection tanks, and rivers by incorporating the Poiger equation, which was effective at predicting the concentrations of EBP in the river waters and wastewaters. Thus, our first trial of verification by field measurements enhanced the practical utility of the Poiger equation, though further study including several photoproducts should be conducted.

Quantitative Distribution of Infectious F-Specific RNA Phage Genotypes in Surface Waters.

UNLABELLED: F-specific RNA phages (FRNAPHs) are considered potential viral indicators of water pollution due to their occurrence and stability in water environments. However, their suitability as viral indicators is not fully elucidated because the characteristics of FRNAPHs are variable depending on the genotype. In this study, for the characterization of infectious FRNAPH genotypes, integrated culture reverse transcription-PCR coupled with the most probable number approach was applied to surface water samples. Further, to recover low concentrations of FRNAPH genotypes, an FRNAPH recovery method was developed. The novel FRNAPH recovery method using a noncharged microfiltration membrane could effectively recover FRNAPH strains without inactivation, while a method using an electronegative microfiltration membrane resulted in the inactivation of some strains. Infectious FRNAPH genotypes in surface water samples were successfully quantified with an efficiency comparable to that of the conventional plaque assay. Genotype I (GI) and GII FRNAPHs tended to be predominant at locations impacted by treated and untreated municipal wastewater, respectively. The numbers and proportions of infectious FRNAPHs tended to be higher during the winter season when water temperature decreased. IMPORTANCE: Properties of FRNAPHs are highly variable depending on their genotypes. Previous typing methods for FRNAPHs are not quantitative and/or are based on molecular assays, which cannot differentiate infective strains from inactive strains. Due to the reasons mentioned above, the utility of FRNAPHs as viral indicators of water pollution has not been fully validated. In this study, a quantitative genotyping method for infectious FRNAPHs was developed and applied to surface water samples. The method enabled characterization of infectious FRNAPH genotypes in terms of their occurrence and seasonality. Moreover, comparison of the method to a conventional molecular assay (reverse transcription-quantitative PCR) enabled characterization of their stability. Our approach can provide novel findings for further validation of FRNAPHs as viral indicators of water pollution.

Detection of physiological activities of G protein-coupled receptor-acting pharmaceuticals in wastewater.

Although pharmaceuticals are generally found at very low levels in aquatic environments, concern about their potential risks to humans and aquatic species has been raised because they are designed to be biologically active. To resolve this concern, we must know whether the biological activity of pharmaceuticals can be detected in waters. Nearly half of all marketed pharmaceuticals act by binding to the G protein-coupled receptors (GPCRs). In this study, we measured the physiological activity of pharmaceuticals in wastewater. We applied the in vitro transforming growth factor-α (TGFα) shedding assay, which accurately and sensitively detect GPCR activation, to investigate the agonistic/antagonistic activities of wastewater extracts against receptors for angiotensin (AT1), dopamine (D2, D4), adrenergic family members (α1B, α2A, ß1, ß3), acetylcholine (M1, M3), cannabinoid (CB1), vasopressin (V1A, V2), histamine (H1, H2, H3), 5-hydroxytryptamine (5-HT1A, 5-HT2C), prostanoid (EP3), and leukotriene (BLT1). As a result, antagonistic activity against AT1, D2, α1B, ß1, M1, M3, H1, and V2 receptors was detected at up to several µg/L for the first time. Agonistic activity against α2A receptor was also detected. The TGFα shedding assay is useful for measuring the physiological activity of GPCR-acting pharmaceuticals in the aquatic environment.