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This is the first report of the detection of two new anti-influenza drugs, peramivir (PER) and laninamivir (LAN), in Japanese sewage effluent and river waters. Over about 1 year from October 2013 to July 2014, including the influenza prevalence season in January and February 2014, we monitored for five anti-influenza drugs-oseltamivir (OS), oseltamivir carboxylate (OC), zanamivir (ZAN), PER, and LAN-in river waters and in sewage effluent flowing into urban rivers of the Yodo River system in Japan. The dynamic profiles of these anti-influenza drugs were synchronized well with that of the numbers of influenza patients treated with the drugs. The highest levels in sewage effluents and river waters were, respectively, 82 and 41 ng/L (OS), 347 and 125 ng/L (OC), 110 and 35 ng/L (ZAN), 64 and 11 ng/L (PER), and 21 and 9 ng/L (LAN). However, application of ozone treatment before discharge from sewage treatment plants was effective in reducing the levels of these anti-influenza drugs in effluent. The effectiveness of the ozone treatment and the drug dependent difference in susceptibility against ozone were further evidenced by ozonation of a STP effluent in a batch reactor. These findings should help to promote further environmental risk assessment of the generation of drug-resistant influenza viruses in aquatic environments.

Background: Tamiflu (oseltamivir phosphate ester, OE) is a widely used antiviral active against influenza A virus. Its active metabolite, oseltamivir carboxylate (OC), is chemically stable and secreted into wastewater treatment plants. OC contamination of natural habitats of waterfowl might induce OC resistance in influenza viruses persistently infecting waterfowl, and lead to transfer of OC-resistance from avian to human influenza. The aim of this study was to evaluate whether such has occurred., Methods: A genomics approach including phylogenetic analysis and probability calculations for homologous recombination was applied on altogether 19,755 neuraminidase (N1 and N2) genes from virus sampled in humans and birds, with and without resistance mutations., Results: No evidence for transfer of OE resistance mutations from avian to human N genes was obtained, and events suggesting recombination between human and avian influenza virus variants could not be traced in the sequence material studied., Conclusions: The results indicate that resistance in influenza viruses infecting humans is due to the selection pressure posed by the global OE administration in humans rather than transfer from avian influenza A virus strains carrying mutations induced by environmental exposure to OC.

In February 2011, at the peak of an influenza outbreak, we performed a comprehensive analysis of the mass balances of four anti-influenza drugs-oseltamivir (OS), oseltamivir carboxylate (OC), amantadine (AMN), and zanamivir (ZAN)-in the urban area of the Yodo River system. This area includes three main river catchments (the Katsura, Uji, and Kidzu Rivers) and is home to 12 million people, about 10% of Japan's population. Water was sampled at six main rivers and 13 tributary sites and eight sewage treatment plants (STPs). We concluded that the STP effluents were the major sources of the anti-influenza drug load in the Yodo River system (68-94% of total mass fluxes). Extended measurement throughout the Yodo River system further showed only small fluctuations of the ratio of OS to OC from 0.2 to 0.3, suggesting that OS and its metabolite are environmentally stable. The results also clearly showed the importance of reducing the levels of anti-influenza drugs in the water environment by reducing their emission at STPs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Time-dependent dynamics in the concentrations of four anti-influenza drugs (oseltamivir, oseltamivir carboxylate, zanamivir, and amantadine) in environmental waters collected from the Yodo River basin, Japan, were monitored for the first time over a 1 year period (July 2010 to June 2011). The clear, convex dynamic profiles of oseltamivir, oseltamivir carboxylate, and zanamivir during a 3 month seasonal influenza outbreak (January to March 2011) were synchronized well with that of the numbers of influenza patients treated with the drugs. The highest levels in sewage treatment plants (STPs) and river waters were, respectively, 177 and 60 ng/L (oseltamivir), 827 and 288 ng/L (oseltamivir carboxylate), and 30 and 15 ng/L (zanamivir). Fixed levels of amantadine were detectable year-round (100-200 ng/L in the STPs and 10-30 ng/L in river waters). The predicted convex profiles of oseltamivir, oseltamivir carboxylate, and zanamivir in both STPs and river waters were significantly correlated (0.714 < R < 0.932) with the observed values. The profiles were predicted successfully by simple mathematical principles, taking the number of influenza patients, quantities of Tamiflu and Relenza used, dilution by drainwaters passing through STPs, removal rates at STPs, dilution rates in river effluents, and attenuation rates in rivers into consideration.

During the 2009 influenza type A(H1N1) pandemic, the antiviral drug oseltamivir (OP, Tamiflu®) was extensively used for treatment and prophylaxis after recommendation from World Health Organisation (WHO). Previous studies have indicated that the pharmaceutically active metabolite of OP, oseltamivir carboxylate (OC), is not readily degraded in sewage treatment plants (STPs) and therefore will be released into receiving waters in elevated concentrations during a pandemic outbreak of influenza. A method for analyzing OP and OC in wastewater by UPLC-TOF has been developed and validated. This analytical method has been used to study the release of OP and OC from a sewage treatment plant outside Oslo, Norway during the 2009 pandemic. Daily flow-proportional influent and effluent samples from 11 weeks covering the main wave of the influenza pandemic were analyzed, and the observed trend in OP and OC concentrations closely followed the trend in percentage of medical consultations caused by influenza-like illness. Concentrations in wastewater influent were in the range of 5-529 ng/L and 28-1213 ng/L for OP and OC, respectively. Concentration data from the 54 influent/effluent sample sets suggest STP removal in the range of -0.8% to 8% for OP and -14% to 0.6% for OC. Statistical analysis of the data sets was inconclusive in determining a removal rate different from 0., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In this work the photodegradation pathways and rates of oseltamivir ester (OE) and oseltamivir carboxylate (OC) were studied under artificial and natural solar irradiation with the goal of assessing the potential of photolysis as a removal mechanism in aquatic environments. The structures of the photoproducts of OE, elucidated by ultra performance liquid chromatography-quadrupole-time-of-flight-mass spectrometry (UPLC-QToF-MS), were proposed to originate from hydration of the cyclohexene ring (TP330), ester hydrolysis (TP284), a combination thereof (TP302), intramolecular cyclization involving the ester (TP312), and cleavage of the ethylpropoxy side chain (TP226). The only photoproduct detected in case of OC was postulated to correspond to the hydration of the α,β-unsaturated acid (TP302). Under simulated solar irradiation the degradation rate of OC was approximately 10 times slower than that of OE, with half-lives ranging from 48 h in ultrapure water to 12 h in surface water from Sant Joan Despi, Llobregat river. The photodegradation under natural solar irradiation during the season of pandemic Influenza peak incidence was about 150 days for OC and 15 days for OE. In general, the photoproducts proved to be more resistant toward further photodegradation than the parent antivirals. In a monitoring survey of surface waters from the Ebro river (NE Spain), OC and OE were detected along with the photoproducts TP330 and 312.

The 2009-2010 influenza pandemic saw many people treated with antivirals and antibiotics. High proportions of both classes of drugs are excreted and enter wastewater treatment plants (WWTPs) in biologically active forms. To date, there has been no study into the potential for influenza pandemic-scale pharmaceutical use to disrupt WWTP function. Furthermore, there is currently little indication as to whether WWTP microbial consortia can degrade antiviral neuraminidase inhibitors when exposed to pandemic-scale doses. In this study, we exposed an aerobic granular sludge sequencing batch reactor, operated for enhanced biological phosphorus removal (EBPR), to a simulated influenza-pandemic dosing of antibiotics and antivirals for 8 weeks. We monitored the removal of the active form of Tamiflu(®), oseltamivir carboxylate (OC), bacterial community structure, granule structure and changes in EBPR and nitrification performance. There was little removal of OC by sludge and no evidence that the activated sludge community adapted to degrade OC. There was evidence of changes to the bacterial community structure and disruption to EBPR and nitrification during and after high-OC dosing. This work highlights the potential for the antiviral contamination of receiving waters and indicates the risk of destabilizing WWTP microbial consortia as a result of high concentrations of bioactive pharmaceuticals during an influenza pandemic., (© 2010 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

We investigated the occurrence and fate of the two antiviral drugs oseltamivir carboxylate (OC)-the active metabolite of Tamiflu-and amantadine (AMT) at three sewage treatment plants (STPs) during the 2008-2009 and 2009-2010 influenza seasons in Japan. Both compounds were detected in all samples analyzed. The concentrations in raw influents at the STPs ranged from 140 to 460 ng L(-1) OC and from 184 to 538 ng L(-1) AMT. Primary treatment gave no substantial removal of the drugs (OC, 2-9%; AMT, 7-17%). Biological nutrient-removal-based secondary treatment (anoxic-oxic-anoxic-oxic and anaerobic-anoxic-oxic) removed 20-37% of OC, whereas extended-aeration-based conventional activated sludge treatment removed <20%. STPs using primary plus biological secondary treatment removed <50% of the drugs. The incorporation of tertiary treatment by ozonation removed >90%. Ozonation after secondary treatment in STPs will be necessary during an influenza pandemic to reduce the risks associated with the widespread use of antiviral drugs., (Copyright (c) 2010 Elsevier Ltd. All rights reserved.)

Tamiflu (oseltamivir ethylester) is an antiviral agent for the treatment of influenza A and B. The pro-drug Tamiflu is converted in the human body to the pharmacologically active metabolite, oseltamivir acid, with a yield of 75%. Oseltamivir acid is indirectly photodegradable and slowly biodegradable in sewage works and sediment/water systems. A previous environmental risk assessment has concluded that there is no bioaccumulation potential of either of the compounds. However, little was known about the ecotoxicity of the metabolite. Ester hydrolysis typically reduces the hydrophobicity and thus the toxicity of a compound. In this case, a zwitterionic, but overall neutral species is formed from the charged parent compound. If the speciation and predicted partitioning into biological membranes is considered, the metabolite may have a relevant contribution to the overall toxicity. These theoretical considerations triggered a study to investigate the toxicity of oseltamivir acid (OA), alone and in binary mixtures with its parent compound oseltamivir ethylester (OE). OE and OA were found to be baseline toxicants in the bioluminescence inhibition test with Vibrio fischeri. Their mixture effect lay between predictions for concentration addition and independent action for the mixture ratio excreted in urine and nine additional mixture ratios of OE and OA. In contrast, OE was an order of magnitude more toxic than OA towards algae, with a more pronounced effect when the direct inhibition of photosystem II was used as toxicity endpoint opposed to the 24h growth rate endpoint. The binary mixtures in this assay yielded experimental mixture effects that agreed with predictions for independent action. This is consistent with the finding that OE exhibits slightly enhanced toxicity, while OA acts as baseline toxicant. Therefore, with respect to mixture classification, the two compounds can be considered as acting according to different modes of toxic action, although there are indications that the difference is a toxicokinetic effect, not a true difference of mechanism of toxicity. The general mixture results illustrate the need to consider the role of metabolites in the risk assessment of pharmaceuticals. However, in the concentration ratio of parent to metabolite excreted by humans, the experimental results confirm that the active metabolite does not significantly contribute to the risk quotient of the mixture., (2009 Elsevier B.V. All rights reserved.)

Background: Oseltamivir phosphate (OP; Tamiflu) is a prodrug of the anti-influenza neuraminidase inhibitor oseltamivir carboxylate (OC) and has been developed for the treatment and prevention of both A and B strains of influenza. The recent increase in OP resistance in influenza A virus (H1N1; commonly called "swine flu") has raised questions about the widespread use of Tamiflu in seasonal epidemics and the potential ecotoxicologic risk associated with its use in the event of a pandemic., Objectives: The objectives of this study were to develop an analytical method for quantitative determination of OC in sewage treatment plant (STP) effluent and receiving river water, and to investigate the occurrence of OC in STP effluent and river water in Japan during a seasonal flu outbreak., Methods: We developed an analytical method based on solid-phase extraction followed by liquid chromatography-tandem mass spectrometry. Using this method, we analyzed samples from three sampling campaigns conducted during the 2008-2009 flu season in Kyoto City, Japan., Results: The highest concentration of OC detected in STP discharge was 293.3 ng/L from a conventional activated-sludge-based STP; however, we detected only 37.9 ng/L from an advanced STP with ozonation as a tertiary treatment. In the receiving river water samples, we detected 6.6-190.2 ng/L OC, during the peak of the flu season., Conclusion: OC is present in STP effluent and river water only during the flu season. Ozonation as tertiary treatment in STP will substantially reduce the OC load in STP effluent during an influenza epidemic or pandemic.

The antiviral drug Tamiflu (Oseltamivir Phosphate, OP), has been indicated by the World Health Organization as a first-line defense in case of an avian influenza pandemic. Recent studies have demonstrated that Oseltamivir Carboxylate (OC), the active metabolite of the prodrug OP, has the potential to be released into water bodies. The present laboratory study focused on basic processes governing the environmental fate of OC in surface water from two contrasting aquatic ecosystems of northern Italy, the River Po and the Venice lagoon. Results of this study confirmed the potential of OC to persist in surface water. However, addition of 5% of sediments resulted in rapid OC degradation. Estimated half-life of OC in water/sediment of the River Po was 15 days. After three weeks of incubation at 20 degrees C, more than 8% of (14)C-OC evolved as (14)CO(2) from water/sediment samples of the River Po and Venice lagoon. At the end of the 21-day incubation period, more than 65% of the (14)C-residues were recovered from the liquid phase of both Po and Venice water/sediment samples. OC was moderately retained onto coarse sediments from the two sites. In water/sediment samples of the River Po and Venice lagoon treated with (14)C-OC, more than 30% of the (14)C-residues remained water-extractable after three weeks of incubation. The low affinity of OC to sediments suggests that presence of sediments would not reduce its bioavailability to microbial degradation.