Detection of the Antiviral Drug Oseltamivir in Aquatic Environments
Identifieur interne : 000A91 ( Pmc/Corpus ); précédent : 000A90; suivant : 000A92Detection of the Antiviral Drug Oseltamivir in Aquatic Environments
Auteurs : Hanna Söderström ; Josef D. J Rhult ; Björn Olsen ; Richard H. Lindberg ; Hiroaki Tanaka ; Jerker FickSource :
- PLoS ONE [ 1932-6203 ] ; 2009.
Abstract
Oseltamivir (Tamiflu®) is the most important antiviral drug available and a cornerstone in the defence against a future influenza pandemic. Recent publications have shown that the active metabolite, oseltamivir carboxylate (OC), is not degraded in sewage treatment plants and is also persistent in aquatic environments. This implies that OC will be present in aquatic environments in areas where oseltamivir is prescribed to patients for therapeutic use. The country where oseltamivir is used most is Japan, where it is used to treat seasonal flu. We measured the levels of OC in water samples from the Yodo River system in the Kyoto and Osaka prefectures, Japan, taken before and during the flu-season 2007/8. No OC was detected before the flu-season but 2–58 ng L−1 was detected in the samples taken during the flu season. This study shows, for the first time, that low levels of oseltamivir can be found in the aquatic environment. Therefore the natural reservoir of influenza virus, dabbling ducks, is exposed to oseltamivir, which could promote the evolution of viral resistance.
Url:
DOI: 10.1371/journal.pone.0006064
PubMed: 19557131
PubMed Central: 2699036
Links to Exploration step
PMC:2699036Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Detection of the Antiviral Drug Oseltamivir in Aquatic Environments</title><author><name sortKey="Soderstrom, Hanna" sort="Soderstrom, Hanna" uniqKey="Soderstrom H" first="Hanna" last="Söderström">Hanna Söderström</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Chemistry, Umeå University, Umeå, Sweden</addr-line></nlm:aff></affiliation></author><author><name sortKey="J Rhult, Josef D" sort="J Rhult, Josef D" uniqKey="J Rhult J" first="Josef D." last="J Rhult">Josef D. J Rhult</name><affiliation><nlm:aff id="aff2"><addr-line>Section of Infectious Diseases, Department of Clinical Sciences, Uppsala University Hospital, Uppsala, Sweden</addr-line></nlm:aff></affiliation></author><author><name sortKey="Olsen, Bjorn" sort="Olsen, Bjorn" uniqKey="Olsen B" first="Björn" last="Olsen">Björn Olsen</name><affiliation><nlm:aff id="aff2"><addr-line>Section of Infectious Diseases, Department of Clinical Sciences, Uppsala University Hospital, Uppsala, Sweden</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"><addr-line>Section for Zoonotic Ecology and Epidemiology, Kalmar University, Kalmar, Sweden</addr-line></nlm:aff></affiliation></author><author><name sortKey="Lindberg, Richard H" sort="Lindberg, Richard H" uniqKey="Lindberg R" first="Richard H." last="Lindberg">Richard H. Lindberg</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Chemistry, Umeå University, Umeå, Sweden</addr-line></nlm:aff></affiliation></author><author><name sortKey="Tanaka, Hiroaki" sort="Tanaka, Hiroaki" uniqKey="Tanaka H" first="Hiroaki" last="Tanaka">Hiroaki Tanaka</name><affiliation><nlm:aff id="aff4"><addr-line>Research Center of Environmental Quality Management, Kyoto University, Otsu, Japan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Fick, Jerker" sort="Fick, Jerker" uniqKey="Fick J" first="Jerker" last="Fick">Jerker Fick</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Chemistry, Umeå University, Umeå, Sweden</addr-line></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">19557131</idno><idno type="pmc">2699036</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2699036</idno><idno type="RBID">PMC:2699036</idno><idno type="doi">10.1371/journal.pone.0006064</idno><date when="2009">2009</date><idno type="wicri:Area/Pmc/Corpus">000A91</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000A91</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Detection of the Antiviral Drug Oseltamivir in Aquatic Environments</title><author><name sortKey="Soderstrom, Hanna" sort="Soderstrom, Hanna" uniqKey="Soderstrom H" first="Hanna" last="Söderström">Hanna Söderström</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Chemistry, Umeå University, Umeå, Sweden</addr-line></nlm:aff></affiliation></author><author><name sortKey="J Rhult, Josef D" sort="J Rhult, Josef D" uniqKey="J Rhult J" first="Josef D." last="J Rhult">Josef D. J Rhult</name><affiliation><nlm:aff id="aff2"><addr-line>Section of Infectious Diseases, Department of Clinical Sciences, Uppsala University Hospital, Uppsala, Sweden</addr-line></nlm:aff></affiliation></author><author><name sortKey="Olsen, Bjorn" sort="Olsen, Bjorn" uniqKey="Olsen B" first="Björn" last="Olsen">Björn Olsen</name><affiliation><nlm:aff id="aff2"><addr-line>Section of Infectious Diseases, Department of Clinical Sciences, Uppsala University Hospital, Uppsala, Sweden</addr-line></nlm:aff></affiliation><affiliation><nlm:aff id="aff3"><addr-line>Section for Zoonotic Ecology and Epidemiology, Kalmar University, Kalmar, Sweden</addr-line></nlm:aff></affiliation></author><author><name sortKey="Lindberg, Richard H" sort="Lindberg, Richard H" uniqKey="Lindberg R" first="Richard H." last="Lindberg">Richard H. Lindberg</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Chemistry, Umeå University, Umeå, Sweden</addr-line></nlm:aff></affiliation></author><author><name sortKey="Tanaka, Hiroaki" sort="Tanaka, Hiroaki" uniqKey="Tanaka H" first="Hiroaki" last="Tanaka">Hiroaki Tanaka</name><affiliation><nlm:aff id="aff4"><addr-line>Research Center of Environmental Quality Management, Kyoto University, Otsu, Japan</addr-line></nlm:aff></affiliation></author><author><name sortKey="Fick, Jerker" sort="Fick, Jerker" uniqKey="Fick J" first="Jerker" last="Fick">Jerker Fick</name><affiliation><nlm:aff id="aff1"><addr-line>Department of Chemistry, Umeå University, Umeå, Sweden</addr-line></nlm:aff></affiliation></author></analytic><series><title level="j">PLoS ONE</title><idno type="eISSN">1932-6203</idno><imprint><date when="2009">2009</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p>Oseltamivir (Tamiflu®) is the most important antiviral drug available and a cornerstone in the defence against a future influenza pandemic. Recent publications have shown that the active metabolite, oseltamivir carboxylate (OC), is not degraded in sewage treatment plants and is also persistent in aquatic environments. This implies that OC will be present in aquatic environments in areas where oseltamivir is prescribed to patients for therapeutic use. The country where oseltamivir is used most is Japan, where it is used to treat seasonal flu. We measured the levels of OC in water samples from the Yodo River system in the Kyoto and Osaka prefectures, Japan, taken before and during the flu-season 2007/8. No OC was detected before the flu-season but 2–58 ng L<sup>−1</sup> was detected in the samples taken during the flu season. This study shows, for the first time, that low levels of oseltamivir can be found in the aquatic environment. Therefore the natural reservoir of influenza virus, dabbling ducks, is exposed to oseltamivir, which could promote the evolution of viral resistance.</p></div></front><back><div1 type="bibliography"><listBibl><biblStruct></biblStruct><biblStruct><analytic><author><name sortKey="Olsen, B" uniqKey="Olsen B">B Olsen</name></author><author><name sortKey="Munster, Vj" uniqKey="Munster V">VJ Munster</name></author><author><name sortKey="Wallensten, A" uniqKey="Wallensten A">A Wallensten</name></author><author><name sortKey="Waldenstrom, J" uniqKey="Waldenstrom J">J Waldenstrom</name></author><author><name sortKey="Osterhaus, Adme" uniqKey="Osterhaus A">ADME Osterhaus</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Webster, Rg" uniqKey="Webster R">RG Webster</name></author><author><name sortKey="Bean, Wj" uniqKey="Bean W">WJ Bean</name></author><author><name sortKey="Gorman, Ot" uniqKey="Gorman O">OT Gorman</name></author><author><name sortKey="Chambers, 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Accinelli</name></author><author><name sortKey="Caracciolo, Ab" uniqKey="Caracciolo A">AB Caracciolo</name></author><author><name sortKey="Grenni, P" uniqKey="Grenni P">P Grenni</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Sacca, Ml" uniqKey="Sacca M">ML Saccà</name></author><author><name sortKey="Accinelli, C" uniqKey="Accinelli C">C Accinelli</name></author><author><name sortKey="Fick, J" uniqKey="Fick J">J Fick</name></author><author><name sortKey="Lindberg, R" uniqKey="Lindberg R">R Lindberg</name></author><author><name sortKey="Olsen, B" uniqKey="Olsen B">B Olsen</name></author></analytic></biblStruct><biblStruct><analytic><author><name sortKey="Singer, Ac" uniqKey="Singer A">AC Singer</name></author><author><name sortKey="Miles, An" uniqKey="Miles A">AN Miles</name></author><author><name sortKey="Gould, Ea" uniqKey="Gould E">EA Gould</name></author><author><name sortKey="Johnson, Ac" uniqKey="Johnson A">AC 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<front><journal-meta><journal-id journal-id-type="nlm-ta">PLoS One</journal-id><journal-id journal-id-type="iso-abbrev">PLoS ONE</journal-id><journal-id journal-id-type="publisher-id">plos</journal-id><journal-id journal-id-type="pmc">plosone</journal-id><journal-title-group><journal-title>PLoS ONE</journal-title></journal-title-group><issn pub-type="epub">1932-6203</issn><publisher><publisher-name>Public Library of Science</publisher-name><publisher-loc>San Francisco, USA</publisher-loc></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">19557131</article-id><article-id pub-id-type="pmc">2699036</article-id><article-id pub-id-type="publisher-id">09-PONE-RA-08337R1</article-id><article-id pub-id-type="doi">10.1371/journal.pone.0006064</article-id><article-categories><subj-group subj-group-type="heading"><subject>Research Article</subject></subj-group><subj-group subj-group-type="Discipline"><subject>Chemistry</subject><subject>Chemistry/Applied Chemistry</subject><subject>Virology/Antivirals, including Modes of Action and Resistance</subject></subj-group></article-categories><title-group><article-title>Detection of the Antiviral Drug Oseltamivir in Aquatic Environments</article-title><alt-title alt-title-type="running-head">Tamiflu in Surface Water</alt-title></title-group><contrib-group><contrib contrib-type="author"><name><surname>Söderström</surname><given-names>Hanna</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Järhult</surname><given-names>Josef D.</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author"><name><surname>Olsen</surname><given-names>Björn</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author"><name><surname>Lindberg</surname><given-names>Richard H.</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author"><name><surname>Tanaka</surname><given-names>Hiroaki</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author"><name><surname>Fick</surname><given-names>Jerker</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib></contrib-group><aff id="aff1"><label>1</label><addr-line>Department of Chemistry, Umeå University, Umeå, Sweden</addr-line></aff><aff id="aff2"><label>2</label><addr-line>Section of Infectious Diseases, Department of Clinical Sciences, Uppsala University Hospital, Uppsala, Sweden</addr-line></aff><aff id="aff3"><label>3</label><addr-line>Section for Zoonotic Ecology and Epidemiology, Kalmar University, Kalmar, Sweden</addr-line></aff><aff id="aff4"><label>4</label><addr-line>Research Center of Environmental Quality Management, Kyoto University, Otsu, Japan</addr-line></aff><contrib-group><contrib contrib-type="editor"><name><surname>Ratna</surname><given-names>Banahalli</given-names></name><role>Editor</role><xref ref-type="aff" rid="edit1"></xref></contrib></contrib-group><aff id="edit1">Naval Research Laboratory, United States of America</aff><author-notes><corresp id="cor1">* E-mail: <email>jerker.fick@chem.umu.se</email></corresp><fn fn-type="con"><p>Conceived and designed the experiments: BO JF. Performed the experiments: HS RHL JF. Analyzed the data: HS JJ BO RHL JF. Contributed reagents/materials/analysis tools: HT. Wrote the paper: HS JJ BO HT JF. Conducted the sampling: HS JJ.</p></fn></author-notes><pub-date pub-type="collection"><year>2009</year></pub-date><pub-date pub-type="epub"><day>26</day><month>6</month><year>2009</year></pub-date><volume>4</volume><issue>6</issue><elocation-id>e6064</elocation-id><history><date date-type="received"><day>22</day><month>1</month><year>2009</year></date><date date-type="accepted"><day>26</day><month>5</month><year>2009</year></date></history><permissions><copyright-statement>Söderström et al.</copyright-statement><copyright-year>2009</copyright-year><license xlink:href="http://creativecommons.org/licenses/by/4.0/"><license-p>This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.</license-p></license></permissions><abstract><p>Oseltamivir (Tamiflu®) is the most important antiviral drug available and a cornerstone in the defence against a future influenza pandemic. Recent publications have shown that the active metabolite, oseltamivir carboxylate (OC), is not degraded in sewage treatment plants and is also persistent in aquatic environments. This implies that OC will be present in aquatic environments in areas where oseltamivir is prescribed to patients for therapeutic use. The country where oseltamivir is used most is Japan, where it is used to treat seasonal flu. We measured the levels of OC in water samples from the Yodo River system in the Kyoto and Osaka prefectures, Japan, taken before and during the flu-season 2007/8. No OC was detected before the flu-season but 2–58 ng L<sup>−1</sup> was detected in the samples taken during the flu season. This study shows, for the first time, that low levels of oseltamivir can be found in the aquatic environment. Therefore the natural reservoir of influenza virus, dabbling ducks, is exposed to oseltamivir, which could promote the evolution of viral resistance.</p></abstract><counts><page-count count="4"></page-count></counts></article-meta></front><body><sec id="s1"><title>Introduction</title><p>Oseltamivir (Tamiflu®) is the most important of the few antiviral drugs available for treatment of seasonal flu and a cornerstone in the defense against a future influenza pandemic. Most governments have built their preparedness plans around stockpiling oseltamivir and it is recommended by WHO both as treatment and prophylaxis in a pandemic situation <xref rid="pone.0006064-WHO1" ref-type="bibr">[1]</xref>.</p><p>Influenza A virus is a zoonosis, with its natural reservoir in dabbling ducks <xref rid="pone.0006064-Olsen1" ref-type="bibr">[2]</xref>. It belongs to the <italic>orthomyxoviridae</italic>, a negative strand RNA virus family, which also includes influenza B and C; however the two latter are of less importance as human pathogens. The different influenza A virus subtypes are named after the type of two cell-surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA) <xref rid="pone.0006064-Webster1" ref-type="bibr">[3]</xref>. At present, there are two subtypes, H1N1 and H3N2, which cause the annual seasonal influenza epidemics <xref rid="pone.0006064-Webster1" ref-type="bibr">[3]</xref>. All influenza infections render humoral immunologic memory, but antigenic changes are so frequent that previous infections often give only limited immunity to concurrent virus. This may be a viral strategy, where the low specificity of the virus polymerase generates frequent mismatches and a high rate of mutations. The influenza virus has another, more drastic way of genetic change, where genetic elements from two viruses infecting the same cell can be recombined. This process, termed ‘genetic reassortment’, promotes rapid evolutionary changes and is the key to the genesis of new strains of human influenza capable of causing a pandemic <xref rid="pone.0006064-Webster1" ref-type="bibr">[3]</xref>–<xref rid="pone.0006064-Lipatov1" ref-type="bibr">[5]</xref>. Both the ‘Asian flu’ in 1957 (H2N2) and the ‘Hong Kong flu’ in 1968 (H3N2) were reassortments between human-adapted seasonal influenza strains and contemporary avian strains <xref rid="pone.0006064-Kawaoka1" ref-type="bibr">[6]</xref>.</p><p>Oseltamivir is a neuraminidase inhibitor administered orally as a prodrug, oseltamivir phosphate, which is converted to the active metabolite OC in the liver and then excreted without further metabolism through the urine <xref rid="pone.0006064-Sweetman1" ref-type="bibr">[7]</xref>. In a previous study, we have shown that the active metabolite of oseltamivir, oseltamivir carboxylate (OC) is neither degraded nor removed in sewage treatment plants (STPs) <xref rid="pone.0006064-Fick1" ref-type="bibr">[8]</xref>. Thus, we assumed that OC can be present in the aquatic environment. Recent publications show that OC is quite persistent in aquatic environments and is only removed by microbial degradation associated with sediment <xref rid="pone.0006064-Accinelli1" ref-type="bibr">[9]</xref>, <xref rid="pone.0006064-Sacc1" ref-type="bibr">[10]</xref>. A hypothesis has been presented that OC residues in the environment, either after usage during a pandemic or for treatment of seasonal influenza, could expose the natural reservoir of influenza virus, dabbling ducks, to low levels of this antiviral which could promote resistance development <xref rid="pone.0006064-Fick1" ref-type="bibr">[8]</xref>, <xref rid="pone.0006064-Singer1" ref-type="bibr">[11]</xref>. OC usage during a pandemic could also have other ecotoxicological effects <xref rid="pone.0006064-Singer2" ref-type="bibr">[12]</xref>.</p><p>Japan is the top per-capita-consumer of oseltamivir; the manufacturer Roche estimates that 6 of 16 million infected Japanese were prescribed the drug during the 2004/05 flu season <xref rid="pone.0006064-F.HoffmannLa1" ref-type="bibr">[13]</xref>. A study was conducted with the assumption that a) OC is detectable in Japanese waterways, b) levels increase at the peak of the flu season and c) levels are higher closer to the outlet of a STP. We chose to study the Yodo River system in the Kyoto and Osaka prefectures, a densely populated area located off-sea.</p></sec><sec id="s2"><title>Materials and Methods</title><sec id="s2a"><title>Sampling</title><p>Surface water was collected before (June 2007) and during (December 2007 and February 2008) the flu season 2007/8 from totally six sites (R1–R6) in three rivers of the Yodo River system (<xref ref-type="fig" rid="pone-0006064-g001">Fig. 1</xref>). R1 (N17° 32.396 E78° 14.590, Kyoto), R2 (N17° 34.422 E78° 21.359, Kyoto), R3 (N17° 34.422 E78° 21.359, Kyoto), R4 (N17° 34.451 E78° 21.390, Kyoto), R5 (N17° 33.165 E78° 19.990, Osaka) and R6 (N17° 36.845 E78° 11.598, Osaka). Repeated sampling was performed at sites R1, R2 and R4. R1 was located in a rural area of Kamo River, before the river enters more populated areas. In Katsura River samples were taken 1 km upstream (R2) and 2.5 km downstream (R4) of the outlet of one of the major STPs in Kyoto prefecture. In February 2008, additional sampling was performed closer to the STP (R3), 0.5 km downstream of its outlet, and in the Yodo River (R5 and R6) in the Osaka prefecture, respectively, where the water has run through almost all of the river system, collecting the sewage outlets from approximately 5 million inhabitants. Duplicate 500 ml samples were taken at each location; these were immediately frozen and kept at −18°C until analysis.</p><fig id="pone-0006064-g001" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0006064.g001</object-id><label>Figure 1</label><caption><title>Sampling locations in Kyoto (R1–R4) and Osaka (R5–R6) prefectures, Japan.</title></caption><graphic xlink:href="pone.0006064.g001"></graphic></fig></sec><sec id="s2b"><title>Chemicals</title><p>Oseltamivir carboxylate (OC), (RO0640802-002; lot: 01007B243804) and Oseltamivir carboxylate labelled with deuterium (OC<sub>D3</sub>), (RO0604802-004; lot: 511-001-2197/4) were obtained from Roche (F. Hoffmann-La Roche Ltd, Basel, Switzerland). Formic acid, ammonium hydroxide 25% and methanol (HPLC-grade) were purchased from JT Baker (Deventer, the Netherlands), acetonitrile (HPLC-grade) from Fischer Chemicals (Zurich, Switzerland) and sulphuric acid from Merck (Darmstadt, Germany). The purified water (resistivity, 18.2 MΩ cm) was prepared by an ELGA MAXIMA HPLC ultra pure water system (ELGA, High Wycombe Bucks, UK). Standard stock solutions of OC and OC<sub>D3</sub>, 100 ng mL<sup>−1</sup>, were prepared in water (10 mL) and kept dark at 4°C.</p></sec><sec id="s2c"><title>Sample pretreatment</title><p>Samples were filtered through 0.45 µm MF™-membrane filters (Millipore, Sundbyberg, Sweden) before acidification to pH 3 using sulphuric acid. The internal standard OC<sub>D3</sub> was added to each sample to an amount of 500 ng. The Strata-X-C (200 mg, 6 mL) mixed mode cation exchange sorbent (Phenomenex, email: <email>international@phenomenex.com</email>) used for the solid phase extraction (SPE) was conditioned and equilibrated by 2.0 ml of methanol and 2.0 ml of deionized water. 500 mL of the samples were applied to the SPE columns at a flow rate of 5 mL min<sup>−1</sup>. Impurities were removed by 2.0 ml 0.1% sulphuric acid and the sorbents were dried (1 min at 10” Hg). Neutral and acidic components were removed by 2 mL of methanol and wasted, followed by elution of the analytes by 2 mL of 5% NH<sub>4</sub>OH in methanol. The eluates were evaporated to approximately 20 µl using air and then reconstituted in acetonitrile in water (1∶1), containing 0.1% formic acid, to a final extract volume of 1.0 ml.</p></sec><sec id="s2d"><title>Liquid Chromatography-Mass Spectrometry</title><p>Sample extracts and calibration solutions (7.5 µl) were injected into a Waters Acquity™ Ultra Performance LC system connected to a Micromass Quattro Ultima triple quadruple mass spectrometer. OC and the internal standard, OC<sub>D3</sub>, were separated by using: equal amounts of the mobile phases H<sub>2</sub>0 and ACN, both containing 0.1% (v/v) formic acid; a flow rate of 0.200 ml min<sup>−1</sup> and a Waters Acquity™ UPLC BEH C18 (2.1×50 mm, 1.7 µm particle size) analytical column. The electrospray was held in positive ion mode and the capillary and cone voltage were kept at 3.9 kV and 45 V, respectively. The source temperature was 110°C and the desolvation temperature was 310°C. The following transitions were monitored 284.9>196.6 (OC) and 287.9>199.9 (OC<sub>D3</sub>). Quantification was based on internal standard calibration and the peak area ratios of OC<sub>D3</sub> and OC <xref rid="pone.0006064-Fick1" ref-type="bibr">[8]</xref>. The linearity of the seven point calibration curve was acceptable (R<sup>2</sup> above 0.997). LOQ was 1 ng L<sup>−1</sup>, determined by using the second point in the calibration curve.</p></sec></sec><sec id="s3"><title>Results</title><p>No OC was detected in the surface water collected in June 2007, during the influenza “off-season” (<xref ref-type="table" rid="pone-0006064-t001">Table 1</xref>) and no influenza patients were reported this week <xref rid="pone.0006064-Kyoto1" ref-type="bibr">[14]</xref>. In December, at the onset of the flu season (<xref ref-type="fig" rid="pone-0006064-g002">Fig. 2</xref>), 2 and 7 ng L<sup>−1</sup> of OC was detected upstream (R2) and downstream (R4), respectively, of the STP (<xref ref-type="table" rid="pone-0006064-t001">Table 1</xref>). At the peak of the flu-season, the levels of OC downstream the STP (R4) increased to 11 and 10 ng L<sup>−1</sup>, and OC levels of 19 ng L<sup>−1</sup> was measured at the additional sampling point (R3) closer to the STP, while the levels of OC detected up-stream of the STP and in Kamo River were similar to those found in December (<xref ref-type="table" rid="pone-0006064-t001">Table 1</xref>). At the peak of the flu-season, the OC concentrations in the Yodo River were also measured and the detected levels were 12 ng L<sup>−1</sup> (R5) and 58 ng L<sup>−1</sup> (R6) (<xref ref-type="table" rid="pone-0006064-t001">Table 1</xref>). In Kyoto City, 6748 individuals were reported to have been affected by the flu during week 7 (11–17th of February) <xref rid="pone.0006064-Kyoto1" ref-type="bibr">[14]</xref> (<xref ref-type="fig" rid="pone-0006064-g002">Fig. 2</xref>), i.e. the week we sampled water at site R2–R6. Assuming that approximately 40% received oseltamivir treatment <xref rid="pone.0006064-F.HoffmannLa1" ref-type="bibr">[13]</xref>, this corresponds to a weekly usage of 1.3 kg OC or 0.18 kg day<sup>−1</sup> (estimated from 30% pediatric dosage, 70% adult dosage and 75% pro-drug conversion to OC, respectively). Calculations from the daily use and the average flow in Katsura River during the 12th of February (2291000 m<sup>3</sup> day<sup>−1</sup>) yield a predicted environmental concentration (PEC) of 78 ng L<sup>−1</sup>. This PEC value is similar to the OC levels that we measured in Katsura River downstream of the STP on the same day. Furthermore, some of the levels detected in this study are close the IC<sub>50</sub> (concentration that causes 50% inhibition) of OC, which however depends heavily on type of virus and exposure system, but such low levels as 80–230 ng L<sup>−1</sup> have been reported <xref rid="pone.0006064-Monto1" ref-type="bibr">[15]</xref>, <xref rid="pone.0006064-Gubareva1" ref-type="bibr">[16]</xref>.</p><fig id="pone-0006064-g002" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0006064.g002</object-id><label>Figure 2</label><caption><title>Number of individuals in Kyoto city and Kyoto prefecture affected by the flu during the flu season 07/08 <xref rid="pone.0006064-Kyoto1" ref-type="bibr">[14]</xref>.</title><p>Arrows indicate sampling events.</p></caption><graphic xlink:href="pone.0006064.g002"></graphic></fig><table-wrap id="pone-0006064-t001" position="float"><object-id pub-id-type="doi">10.1371/journal.pone.0006064.t001</object-id><label>Table 1</label><caption><title>Average water concentrations (ng L<sup>−1</sup>) of oseltamivir carboxylate (OC) measured in Kyoto (R1–R4) and Osaka (R5–R6) prefectures, Japan.</title></caption><alternatives><graphic id="pone-0006064-t001-1" xlink:href="pone.0006064.t001"></graphic><table frame="hsides" rules="groups"><colgroup span="1"><col align="left" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col><col align="center" span="1"></col></colgroup><thead><tr><td align="left" rowspan="1" colspan="1">Date</td><td align="left" rowspan="1" colspan="1">Week</td><td align="left" rowspan="1" colspan="1">Kamo River</td><td colspan="3" align="left" rowspan="1">Katsura River</td><td colspan="2" align="left" rowspan="1">Yodo River</td></tr><tr><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">R1</td><td align="left" rowspan="1" colspan="1">R2</td><td align="left" rowspan="1" colspan="1">R3</td><td align="left" rowspan="1" colspan="1">R4</td><td align="left" rowspan="1" colspan="1">R5</td><td align="left" rowspan="1" colspan="1">R6</td></tr></thead><tbody><tr><td align="left" rowspan="1" colspan="1">2007-06-13</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1">nd</td><td align="left" rowspan="1" colspan="1">nd</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">nd</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">-</td></tr><tr><td align="left" rowspan="1" colspan="1">2007-12-17</td><td align="left" rowspan="1" colspan="1">50</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">7</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">-</td></tr><tr><td align="left" rowspan="1" colspan="1">2008-02-04</td><td align="left" rowspan="1" colspan="1">5</td><td align="left" rowspan="1" colspan="1">2</td><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">11</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">-</td></tr><tr><td align="left" rowspan="1" colspan="1">2008-02-12</td><td align="left" rowspan="1" colspan="1">6</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">4</td><td align="left" rowspan="1" colspan="1">19</td><td align="left" rowspan="1" colspan="1">10</td><td align="left" rowspan="1" colspan="1"></td><td align="left" rowspan="1" colspan="1"></td></tr><tr><td align="left" rowspan="1" colspan="1">2008-02-15</td><td align="left" rowspan="1" colspan="1">6</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">-</td><td align="left" rowspan="1" colspan="1">12</td><td align="left" rowspan="1" colspan="1">58</td></tr></tbody></table></alternatives></table-wrap></sec><sec id="s4"><title>Discussion</title><p>We found that the active metabolite of oseltamivir is present in Japanese waterways at clearly detectable levels. The levels increase at the peak of the flu season and are in the same magnitude as the PEC value estimated from the total use of oseltamivir, and close to the IC<sub>50</sub> of OC. Our results suggest that OC levels are higher closer to major STPs and further downstream in a river system. Subsequently, the natural reservoir of influenza virus, dabbling ducks <xref rid="pone.0006064-Olsen1" ref-type="bibr">[2]</xref>, living in the aquatic environments in Japan or another area where oseltamivir is used widely, are exposed to OC. As influenza in dabbling ducks is a gastrointestinal infection, their bowel can contain replicating virus as well as oseltamivir, which could promote the evolution of viral resistance.</p><p>Strain-specific vaccines will not be available at the start of a pandemic. The only intervention possible in the absence of vaccines would be the use of antivirals. Earlier pandemic influenza viruses have contained genetic material from avian strains and if oseltamivir-resistance is induced and transferred from the avian reservoir to a virus with pandemic potential there is an imminent risk that one of the cornerstones in pandemic preparedness have been disarmed. It is therefore essential to investigate the biological significance of the observed levels of OC in the environment.</p></sec></body><back><ack><p>We thank F.Hoffmann-La Roche Ltd. for donating oseltamivir carboxylate and oseltamivir carboxylate labelled with deuterium. 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