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Synchronous Dynamics of Observed and Predicted Values of Antiinfluenza drugs in Environmental Waters during a Seasonal Influenza Outbreak Takashi Azuma, Norihide Nakada, Naoyuki Yamashita, and Hiroaki Tanaka* Research Center for Environmental Quality Management, Graduate School of Engineering, Kyoto University, 1-2 Yumihama, Otsu, Shiga 520-0811, Japan S Supporting Information *

ABSTRACT: 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.

1. INTRODUCTION A number of anti-influenza drugs are available in the market for the treatment and prophylaxis of human influenza. In Japan, Tamiflu has been used since 20001 and is the most popular of these drugs; Japan accounts for more than 70% of its world consumption. 2 Tamiflu is a prodrug (oseltamivir, OS, formulated as a phosphate) that is converted into its active metabolite, oseltamivir carboxylate (OC), in the liver.3−5 In addition to Tamiflu, Relenza (zanamivir, ZAN), and Amantadine (AMN) are used as anti-influenza drugs, in Japan. OS and ZAN are recommended by the World Health Organization for both treatment and prophylaxis for mitigation during influenza outbreaks.6 Use of AMN as a remedy for influenza is, however, not recommended;7 like rimantadine it inhibits the biosynthesis of virus M2 protein, but the virus had acquired high levels of resistance to these drugs (70−90% in 2005−20068) through substitutions in this protein. Recently questions have been posed about its safety after release into environmental waters. In the case of Tamiflu, OC is © 2012 American Chemical Society

difficult to break down after discharge from the human body9−15 and both OS and OC were measured in sewage waters at sewage treatment plants (STPs) in a range of 5−1213 ng/L.16−19 The concentrations of OS and OC in river waters were also in a range of 10−865 ng/L.12,18,20−22 These previous investigations indicated that OS and OC posed no significant risk in the water environment, even in a new influenza pandemic,11 in view of a chronic-ecotoxicity-derived predicted no effect concentration of 100 μg/L.11,23−25 The estimated concentration of AMN in STP influent varies from 184 to 538 ng/L,16 but its concentration, fate and eco-safety in river waters are not known. No such information is also available for ZAN. Even in the case of Tamiflu, considerable uncertainties remain regarding risk evaluation methodology and working assumpReceived: Revised: Accepted: Published: 12873

August 7, 2012 October 15, 2012 October 29, 2012 October 29, 2012 dx.doi.org/10.1021/es303203c | Environ. Sci. Technol. 2012, 46, 12873−12881

Environmental Science & Technology

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tions, as pointed out by Ellis in 2010.4 The possible risks of long-term chronic effects and the appearance of new influenza viruses with tolerance to anti-influenza drugs through waterfowl have also been pointed out by several researchers.9,21,26−28 Risk management for problems originating from the release of antiinfluenza drugs into environmental waters still evokes concern during influenza pandemics. Evaluation of the exact amounts of anti-influenza drugs released into the water environment during influenza pandemics or outbreaks is therefore the starting point for finding a solution to these potential problems. Prediction of the concentrations of anti-influenza drugs discharged is required, not only to assess environmental loads in the face of possible hazards but also to simulate environmental distribution or fate after discharge. Several initial trials have been done to estimate predicted environmental concentrations (PECs) of OC in environmental waters in influenza outbreaks.4,11,26,29,30 Singer et al.26,30,31 used a dynamic hydrologic model with working assumptions to estimate the PEC values of OC released into river waters in several US and UK catchments. Their modeling was further extended by Straub (2009)32 to estimate PEC values in surface waters and STPs for the River Lee catchment in the UK and the Lower Colorado basins during both in seasonal and pandemic flu season. Fick et al. (2007)9 proposed a similar equation for estimating PEC of OC and applied it to obtain a PEC surfacewater_Japan of 0.028 μg/L during 2004−2005. Söderstöm et al. (2009)22 calculated a PEC for OC of 78 ng/L in the surface waters of the Katsura river in the Yodo River basin during the influenza season (2007−2008). Ghosh et al. (2010)21 tried to estimate the PECs of OC in Kyoto City STP discharges at three different stages during the influenza outbreak season (2008−2009) to give a value (311 ng/L) close to the maximum detected concentration of OC (293.3 ng/L). In the same year, Ellis (2010)4 obtained PECs for OC (40−80 μg/L) in the surface waters of the River Lee catchment in North East London during an influenza pandemic; they used the values for risk assessment of urban receiving waters. Recently, Leknes et al. (2012)17 predicted the concentration of OS + OC in STP influents in two Norwegian counties, Oslo and Akershus, during the 2009 influenza pandemic to have an average value of 1.9 μg/L, which was similar to the measured concentration. A tentative final goal for this line of investigation is to achieve unambiguous consistency between measured and predicted concentrations throughout the influenza season. Several attempts have further been made to show the timedependent dynamics of influenza drugs released into the water environment during the influenza season.17,21,22,33 Due to a wide range difference in estimated persistent times of the drugs in the human body (6−20 h), discrepancies have been observed between the time-dependent dynamic profile of the measured concentrations and the numbers of influenza patients who were prescribed the drugs.34,35 Back-estimations of the PECs indicated that overestimation had occurred.17 Further verification of the experimental and prediction methodology therefore seems necessary to refine the overall time-based distributions of influenza drugs released into the water environment so that they match the profiles of the measured concentrations. In this study, we examined the time-dependent dynamics of four anti-influenza drugs, OS, OC, ZAN, and AMN, in environmental waters collected from the Yodo River basin in the Kansai area of Japan throughout 1 year (2010−2011), including a seasonal influenza outbreak from January to March 2011. Our emphasis was the dynamics in the concentrations of

these anti-influenza drugs at STPs, because influx of sewage to STPs is the first step in discharge into the environment. Furthermore, we predicted the time-dependent dynamic levels of OS, OC, and ZAN in environmental waters by application of simple mathematical principles and statistically evaluated their accuracy by comparing with the observed values.

2. MATERIALS AND METHODS 2.1. Sampling. During 1 year from July 2010 to June 2011, including the influenza outbreak season from January to March 2011, a total of 146 grab river water samples from each of seven sites and 127 sewage samples (60 influents and 67 effluents) from each of five STPs (as 24 h composite samples) were collected from the Yodo River basin in the Kansai area of Japan. Samplings at STPs were made in the continuous proportional mode as described by Ort et al. (2010).36 The seven river water collection points were located at five river sites (along the Yodo River basin or on the Katsura, Uji, and Kidzu rivers in Kyoto Prefecture, and on the Yodo River in Osaka Prefecture) and two tributaries. The STP sites consisted of a group of four sites where a conventional activated sludge (CAS) process followed by chlorination for disinfection was used, and the remaining site, where ozonation was used for decolorization of dyes after CAS. Information on the STP sites is given in Supporting Information (SI) Figure S1 and Tables S1 and S2. Sampling was done on nonrainy days once a week during the influenza outbreak season and once a month for the rest of the year. No rain was observed during 2 days before the sampling day. Oneliter samples were collected in 1 L g1ass bottles containing ascorbic acid at 1 g/L as a preservative. All water samples were immediately transported to the lab (within 4−6 h) and they were stored 4 °C under darkness and processed within 24 h. 2.2. Chemicals and Reagents. Oseltamivir (purity 98%) (OS: C16H28N2O4, M+ 312.40, pKa 7.737,38) and oseltamivir carboxylate (purity 99%) (OC: C14H24N2O4, M+ 284.35, pKa 3.8 (acid), 7.8 (base)11,39) were purchased from Sigma-Aldrich (St. Louis, MO). Zanamivir (purity 98%) (ZAN: C12H20N4O7, M+ 332.31, pKa 3.8 (acid), 11.3 (base)39) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Amantadine (purity 99%) (AMN: C10H17N, M+ 151.24, pKa 10.140) was purchased from LKT Laboratories (St. Paul, MN). LC-MS-grade solvents (methanol, acetone, and acetonitrile), formic acid, hydrochloric acid, ascorbic acid, and triethylamine were purchased from Wako Pure Chemical Industries, Ltd. (Japan). Individual standard stock solutions of OS, OC, ZAN, and AMN at 1 mg/100 mL were prepared in methanol and stored at −30 °C. 2.3. Analytical Procedure. Samples from river water and STP influents and effluents were separately filtered through glass fiber filters (GF/B, 1-μm pore size, Whatman, Maidstone, UK); each filtrate was divided into two 30 mL portions for river and STP effluent and 50 mL for STP influent. The two aliquots were concentrated and analyzed with and without addition of the appropriate standard stock solution for recovery correction. Only filtered water samples were used for analysis, because the distribution coefficients of OS, ZAN, and AMN between noctanol and water (logKOW) are lower than 2.5,41 which is the threshold for adsorption (0.41 for OS,38 −7 for ZAN,24 and 2.4 for AMN;42 drug adsorption on sewage sludge was negligibly small or close to 0% in the case of OC.9 The sample solution was then analyzed by combination of a strong-cation solid phase extraction cartridge (Bond Elut SCX (500 mg), Agilent Technologies, Santa Clara, CA; elution solvent, 10% triethyl12874

dx.doi.org/10.1021/es303203c | Environ. Sci. Technol. 2012, 46, 12873−12881


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Figure 1. Time-dependent dynamics of OS (a), OC (b), ZAN (c), and AMN (d) concentrations in the influents of STPs (S1−S5) from July to June 2010−2011, including the influenza outbreak season (January to March 2011).

quantified (NQ), respectively. Recovery rates for OS, OC, ZAN, and AMN from river waters (n = 7) were in the ranges of 61.6 ± 1.4, 66.2 ± 8.1, 39.2 ± 8.1, and 75.4 ± 1.0%, respectively. The values for STP effluents (n = 5) were 61.6 ± 1.4, 66.2 ± 8.1, 39.2 ± 8.1, and 75.4 ± 1.0% for OS, OC, ZAN, and AMN; for STP influents (n = 5) they were 36.4 ± 4.9, 62.7 ± 2.0, 23.3 ± 8.2, and 62.9 ± 1.0%, respectively. Furthermore, the values of reproducibility assessed by injection of river water or STP effluent or influent spiked at 50 ng/L (n = 3) were in the ranges of 1.4−8.0, 1.4−2.0, 5.4−8.2, and 1.0−1.3% for OS, OC, ZAN, and AMN, respectively. 2.5. LC-MS/MS Condition. OS, OC, ZAN, and AMN were separated in a UPLC BEH C18 column (2.1 mm ×100 mm, 1.7 μm, Waters Co., Milford, MA) fitted to a binary pump system (Waters Acquity Ultra Performance LC, UPLC). A gradient elution program was achieved at 60 °C with a mixed solvent system of 0.1% formic acid (v/v) in water (A) and acetonitrile (B) at a flow rate of 0.35 μL/min under a program of 0.00− 4.00 min (5% B), 4.00−4.30 min (25% B), 4.30−5.80 min (80% B), 5.80−6.00 min (80% B); return to the starting solvent was achieved by elution with 5% B for 6.00−8.00 min to condition the column. The UPLC system was coupled to a Quattro Micro API MS (Waters) equipped with an electrospray ionization source and interface, and it was operated in positive ion mode. Product ions were generated with collision energies of 10, 10, 10, and 14 eV for OS, OC, ZAN, and AMN, respectively. Instrument control and data acquisition and quantification were performed with Mass Lynx 4.1 software (Waters). Under these conditions the OS, OC, ZAN, and AMN were eluted at 4.92, 3.30, 0.67, and 3.00 min, respectively.

amine in a 1:1 (v/v) mixture of acetone and water) and LCMS/MS. Quantification was performed by using linear calibration curves in the concentration range of 0.5−500 ng/ mL with high reliability (R > 0.999) and linearity (r2 > 0.999). The concentrations of OS, OC, ZAN, and AMN were determined by subtracting the data on the blank solution from the data given by the addition of a known amount of standard solution to cancel out unknown matrix effects.18 2.4. Validation. For quantification of OS, OC, ZAN, and AMN, 7-point calibration was applied in a concentration range from 0.5 to 500 ng/mL in a 4:1 (v/v) mixture of 0.1% formic acid solution: methanol. Linear calibration curves given for these anti-influenza drugs all had r2 > 0.999. Quantification was then performed by using each regression curve. The standard deviation (σ) of the lowest concentration for every anti-influenza drug was calculated on the basis of the data from five independent experiments (n = 5; coefficient of variation was below 5%) and used to calculate the limit of detection (LOD, 3σ) and limit of quantification (LOQ, 10σ).21,43 The LOD and LOQ values of OS, OC, ZAN, and AMN were 0.1, 0.2, 0.4, and 0.1 ng/L and 0.2, 0.7, 1.3, and 0.2 ng/L, respectively. Relative standard deviation (reproducibility values) for OS, OC, ZAN, and AMN, assessed by injecting Milli-Q water spiked at 50 ng/L (n = 3), were 0.8, 2.3, 4.0, and 1.8%, respectively. The corresponding values for the environmental water samples were estimable on the basis of the concentrations at signal-to-noise (S/N) ratios of 3 for LOD and 10 for LOQ, according to the methods used for pharmaceuticals and personal care products (PPCPs)44 and anti-influenza drugs.21 Therefore, data at S/N < 3 for LOD and 3 ≤ S/N < 10 for LOQ were expressed as not detected (ND) and not 12875



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Figure 2. Time-dependent dynamics of OS (a), OC (b), ZAN (c), and AMN (d) in river waters at sites R1−R7 during July to June 2010−2011, including in the influenza outbreak season (January−March, 2011).

3. RESULTS AND DISCUSSION 3.1. Time-Dependent Dynamics of OS, OC, ZAN, and AMN Present in River Water and STP Influents and Effluents. Because entry of wastewater to the environment begins with sewage influent from the STP, dynamics of the concentrations of the anti-influenza drugs in the STP influents were analyzed. Representative results from STPs located in the middle reaches of the Yodo River close to Kyoto (sites SI S1− S5, Tables S1 and S2) are shown in Figure 1, along with the dynamics in the numbers of influenza patients of influenza patients/sentinel/week.45 The standard deviation of discharge volume from all STPs during measurement was limited to within 10% of the mean flow rate (SI Table S3). This indicates the smallness of the contribution of the water flow rates to the concentrations of OS, OC, ZAN, and AMN determined; it also shows the validity of measuring the concentrations of these anti-influenza drugs in environmental waters. The concentrations of OS, OC, and ZAN stayed at the ND level from July to October 2010, began to increase from November by several ng/L synchronously with the increase in the number of influenza patients, and reached a clear peak at the end of January and early February 2011. The maximum concentrations detected in the influent of the STP for OS, OC, and ZAN were 174, 827, and 30 ng/L, respectively. The corresponding peak values in the effluents of the STPs were 177 ng/L (OS), 814 ng/L (OC), and 26 ng/L (ZAN), respectively (SI Figure S2). These peak values were counted within the reported values (5−1213 ng/L).16−19 The present overall results indicated a clear difference in the dynamic profiles between the group including OS, OC, and ZAN, which

showed a synchronized convex increase with spreading of the influenza outbreak, and AMN, which was distributed throughout the year and showed no such clear synchronized variation. The peak concentrations of OS and OC were synchronized well with the numbers of influenza patients. Although ZAN was detected at 2−3 orders of magnitude lower than OC, it was only detected during influenza outbreak season over the LOD. The concentrations of these anti-influenza drugs dropped quickly with termination of the influenza outbreak; the drugs were not detectable in May 2011, after the end of the outbreak. The low concentration of ZAN may have been due to its low dose (20 mg/day)34 in comparison with the high dose (150 mg/day) of Tamiflu.38,46 Synchronization of the number of influenza patients with the concentrations of OS, OC, and ZAN at the STPs was reasonable because about 90% of these drugs are discharged rapidly from patients within 6−20 h after ingestion.34,35 In contrast, AMN was detectable in all seasons throughout the year at 100−200 ng/L in both influents and effluents at all STPs, and it showed no meaningful correlation with the occurrence of influenza (P > 0.05). These results indicate that AMN was not used at higher levels in the influenza outbreak in Japan. The constant detection of AMN may have been due to its everyday use in the treatment of Parkinson’s disease. Comparative analysis of the data from all the STPs (SI Table S4) indicated that the removal rates of OS, OC, and AMN at four STPs (sites S2−S5) where a CAS process followed by chlorination for disinfection operated were as low as 3−14% (OS), 3−16% (OC), and 4−17% (AMN) (mean values). These removal rates fell into the ranges reported in the 12876



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literature: 0−20% for OS19 and OC16,17,47 and 0−30% for AMN.16,47 At STP site S1, where ozonation was used after CAS, the removal rates were as high as 90% and 94% for OS and OC, respectively. The removal rate of AMN was not as high as for OS and OC; it was in the order of 60%. These results show the effectiveness of ozone treatment for removing these anti-influenza drugs from sewage; this is supported by previous reports of the effectiveness of ozone treatment in removing OC and AMN.16,47 Meaningful estimation of the removal rate of ZAN was, however, difficult because ZAN was present at such low levels (ng/L to several tens of ng/L). We then analyzed the time-dependent dynamics of the concentrations of the anti-influenza drugs in river water. Representative results from sites R1-M and R4-M to R7-M, located in the middle reaches of the Yodo River close to Kyoto, are shown in Figure 2, along with the dynamics of the number of influenza patients/sentinel/week, as shown in Figure 1. Because the flow volumes in all rivers during measurement varied within 10−20% of the mean flow volume (SI Table S3), as did the values at all STPs, the concentrations of the antiinfluenza drugs in the river water were determined as indexes of their time-dependent dynamics. OS, OC, and ZAN were not detectable until October 2010; their concentrations then began to increase from November (several ng/L) synchronously with the increase in the number of influenza patients and reached a clear peak during the end of January and early February 2011. The convex time-dependent dynamic profiles observed in the river water coincided well with the profiles at the STPs (Figure 1). At the river sites the highest values of OS and OC at the peak of the influenza outbreak were 60 and 288 ng/L, respectively, which fell into the reported values of 10−865 ng/ L.9,14,17−19 The presence of ZAN at low levels (several ng/L) was observed only around the peak of the influenza outbreak. AMN was detected at 30−110 ng/L throughout the year and did not show any synchronous variation with the influenza outbreak, confirming the difference in dynamic profiles between AMN and OS, OC, and ZAN as a group. Recently Leknes et al. (2011) reported the time trend of release of OS and OC from one STP in Norway during the influenza pandemic season in 2009.17 Our study is the first to show the wide time-dependent dynamics in the concentrations of four anti-influenza drugs, including ZAN and AMN, in the area of the main river basin in the Kansai district of Japan through monitoring for a long time (1 year); the dynamics in the doses of the drugs were highly consistent with the changes in the numbers of influenza patients treated except for AMN. Concentrations of OC have been previously reported in the ranges of 40−293 ng/L for STP effluents and 2−190 ng/L for river waters, with a broad convex distribution profile, in analyses over several time intervals during the influenza season.21,22 We evaluated the new data in comparison with these previous results. Our sampling was done at 1-week intervals, including the peak of the seasonal influenza outbreak. The dynamic of the concentrations of OC detected showed a clear convex profile, ranging from 50−580 ng/L for the STP effluents and N.D. to 290 ng/L for the river waters; these values were well synchronized with the total numbers of influenza patients treated with the drugs. Our results indicated importance of three compoundsOS, OC, and ZANin Japan on account of their entry into the water environment during influenza outbreaks. The ratio of OS to OC is an important factor in investigating the origin of these drugs. OS is converted into its active

metabolite (OC) in the human body and discharged as a mixture of about 15% OS and about 80% OC (a ratio of OS/ OC of about 0.2).3,11,46 However, the ratios of OS/OC in STP effluents and river waters vary in the range of 0.2−2.0.17,18,20 In our study (SI Figure S3) the ratios were close to 0.2, namely 0.20 ± 0.05 (STP influents, n = 62), 0.21 ± 0.05 (STP effluents, n = 67), and 0.21 ± 0.06 (river waters, n = 147). Statistical analyses (t-test) found no significant difference between the ratios in our water samples and the range of values measured at the time of patient excretion,3,11,46 suggesting strongly that the amounts of OS and OC detected in all of our water samples originated from Tamiflu ingested by patients. Furthermore, the constant nature of the OS/OC ratios in the river waters over the 1 year study period indicated the environmental stability of these drugs and negligibility of conversion from OS to OC. The environmental stability of OS and OC was also in accord with the results of experiments on batchwise photodegradation9−12,48 and biodegradation.13−15 We carried out 1 year survey from 2010 July to 2011 June on the concentrations of four anti-influenza drugs, OS, OC, ZAN, and AMN, in environmental waters for the first time that includes the 3 month seasonal influenza outbreak (January to March 2011). It was concluded that the dynamic profiles of the observed data for OS, OC, and ZAN were well synchronized with those of the numbers of influenza patients treated with the drugs. The highest levels of OS, OC, and ZAN in the STP influents were 174, 827, and 30 ng/L, respectively. Similar levels were detected in the STP effluents: 177 ng/L for OS, 814 ng/L for OC, and 26 ng/L for ZAN. In the case of AMN, however, fixed levels were detected throughout the year, without any relationship with influenza: 100−200 ng/L in the STP influents and effluents, and 30−110 ng/L in the river waters. 3.2. Prediction of Concentrations of Anti-Influenza Drugs in STP Influents during Seasonal Influenza Outbreak. We predicted the concentrations of OS, OC, and ZAN in the environmental waters. First, we predicted the concentrations of OS, OC, and ZAN in STP influents during the seasonal influenza outbreak and compared them with the observed data on the basis of the numbers of influenza patients reported by the Infectious Disease Surveillance Center in Japan, as well as the drug amounts, dose rates, and dilution factors. Similarly, we predicted the concentrations of these drugs in river waters and compared them with the observed data by taking into account the removal rates at STPs and the rates of dilution and decrease in river waters. The concentration of Tamiflu (OS and OC) or Relenza (ZAN) in STP influent (C(STP), in ng/L/day) can be calculated by eq 1: C(STP)in =

P flu×D × E × fdc × Tmp × Fmc WSTP

× 1, 000

(1)

where Pflu is the number of influenza patients in a service area of a STP (persons/day); D is the mean dose of Tamiflu or Relenza per day and per influenza patient (taking the age distribution of influenza patients into consideration) (mg/day/ patient); E is the excretion rate of the anti-influenza drug Tamiflu (15% for OS and 80% for OC)3,11,46 or Relenza (80% for ZAN);34,49 fdc is the rate of prescription of Tamiflu (about 40−60% for OS)50−52 or Relenza (about 15−25% for ZAN)52,53 for influenza patients in Japan; Tmp is the medication period factor set as 5 by taking account the number of influenza 12877



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Figure 3. Relationships between measured and predicted concentrations of OS(a), OC(b), and ZAN(c) in STP influents. For prediction the rates of prescription for OS (about 40−60%), and ZAN (about 15−25%) were used. Each predicted concentration is plotted as a vertical line, the range of which varied with the prescription rate of each drug (n = 62); means are represented by colored dots.

Figure 4. Relationships between measured and predicted concentrations of OS(a), OC(b), and ZAN(c) in river waters at site R-7M. For prediction the rates of prescription for OS (about 40−60%), and ZAN (about 15−25%) were used. Data are plotted as shown in Figure 3.

patients treated with anti-influenza drugs for 5 days;11,34,38,46 Fmc is the factor, 0.06,53 which shows percentage of unused anti-influenza drugs after prescription in Japan; WSTP is the amount of influent water at an STP (m3/day); and 1000 is a factor for unit conversion from mg/m3 to ng/L. In addition, no time loss was assumed for the flow of sewage inside sewerage pipes from the position of upstream discharge after treatment with Tamiflu or Relenza to the downstream STP, because more than 90% of the Tamiflu and Relenza taken is excreted within 6−20 h after treatment.34,35 Another attempts have been made to predict the concentrations of anti-influenza drugs in the water environment,4,6,8,13,18,19,26,30,33 but the major problem that has arisen is improvement of the accuracy of the predicted values.34,35 To overcome this problem we improved the method used to estimate the number of influenza patients by using the total numbers of influenza patients/sentinel/week and the mean dose of Tamiflu or Relenza per influenza patient, taking into consideration the age of each patient. Furthermore, we considered the effects of the percentages of unused drug; this calculation was based on the prescription history of these drugs in Japan and their dilution inside the sewerage system in terms of the flow rate at each STP. Pflu values were estimated based on the patient numbers derived from surveillance done by the Infectious Disease Surveillance Centers in Japan.45,54−57 D values were estimated by dividing the doses calculated from the recommendations for Tamiflu (75 mg twice a day for an adult and 2 mg/kg twice a day for a child)38,46 and Relenza (10 mg twice a day for both adults and children) by the numbers of

patients in each age group.45 Mean doses for Tamiflu and Relenza were estimated as 120−130 mg/patient/week and 20 mg/patient/week, respectively (SI Figure S4). Wstp values in m3/day were cited from the amounts of influent water at the 5 STPs.58 On the basis of the conditions presented above, the predicted concentrations of the three anti-influenza drugs in STP influents were analyzed statistically and plotted against the observed concentrations (Figure 3). There were significant positive correlations between all observed concentrations of the three anti-influenza drugs OS, OC, and ZAN in STP influents and their predicted concentrations (R > 0.915 (OS), 0.932 (OC), and 0.714 (ZAN)), with high linearity (r2 > 0.820 (OS), 0.853 (OC), and 0.522 (ZAN)). All regression lines had a slope of 1 and an intercept of 0 in accord with the theoretical values, supporting the validity of the predictions. For such predictions, the number of influenza patients and the amounts of anti-influenza drugs consumed are the two most important factors. Next, we used eq 2 to predict the concentrations of OS, OC, and ZAN in river water by using the STP influent concentrations predicted above, as well as the rate of removal at STPs and river dilution and attenuation factors. The final predicted concentration of an individual anti-influenza drug at a certain river site per day (C(Riv), ng/L/day) can be calculated with one-by-one correction of the effect of every STP located upstream in each river: C(Riv) = C(STP)in × (1 − R STP) × F × (1 − RRiv ) 12878

(2)



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where RSTP is the removal rate at the STP (%); F is the factor for dilution in the river including the contribution of STP(s) located upstream in the river; and RRiv is the factor for attenuation in the river (%). Samples of river water collected at site R-7 M was used as representative for prediction. Site R-7 M was selected because it was located at the most downward of the area monitored. RSTP for OS and OC was set to 10% on the basis of the data in Section 3.1. RSTP for ZAN was also set to 10% because of the similarity of the drug’s physicochemical properties to those of OS and OC.24,38 RRiv was set to 0% for all anti-influenza drugs, because all of the drugs were sufficiently stable in the environmental waters.10 Laboratory-scale batch-wise degradation indicated that the half-life of OC was 30−150 days by photodegradation10,12 and 20−50 days by biodegradation.15 All F values for OS, OC, and ZAN were then set to about 0.1 (0.1 ± 0.05) at R-7 M (SI Table S3) by taking null attenuation for the river RRiv into consideration. Finally, we predicted the concentrations of OS, OC, and ZAN in river waters and evaluated the correlations between the predicted and observed values. There were significant positive correlations for all values of OS (a), OC (b), and ZAN (c) (R > 0.861 (OS), 0.846 (OC), and 0.864 (ZAN); Figure 4), as occurred with the STP influents (Figure 3). The high linearity (r2 > 0.712 (OS), 0.687 (OC), and 0.741 (ZAN)), together with slope 1 and intercept 0 of the regression line for all drugs, also validated our predictions for river waters. The results indicated that it was possible to predict the concentrations of OS, OC, and ZAN in both discharged and river water samples by using the model developed here. The model includes relatively simple mathematical principles. Therefore, by simple changes in the parameters used, the concentrations of OS, OC, and ZAN in the environmental waters during influenza pandemics of varying degrees of severity could be estimable. Moreover, it could be predicted to simulate the effectiveness of measures to increase removal rates in order to reduce environmental risk especially appearance of new influenza viruses with tolerance to antiinfluenza drugs through waterfowl.6,18,26−28 Our prediction model might be useful for estimating the concentrations of OS, OC, and ZAN in diverse environmental waters during future pandemics of new types of influenza. Furthermore, by changing parameters such as removal rates at STPs and dilution factors in rivers, application of this prediction model could make it much easier for administrators of STPs and river waters to estimate the effectiveness of their control countermeasures. Introduction of a new technology such as application of Bioplastic moving bed biofilm carriers proposed by Accinelli et al. (2012)59 will contribute to further enhance the removal rates. In conclusion we successfully predicted the time-dependent dynamic levels of OS, OC, and ZAN in the influents of STPs and river waters and found good positive statistical correlations (significance level P < 0.01) between each set of observed convex-shaped concentration profiles including at the peak of the seasonal influenza outbreak. These significant positive correlations between the predicted and observed values in environmental waters might help in risk management of the environmental effects of discharged OS, OC, and ZAN during new influenza pandemics in future.

Article

ASSOCIATED CONTENT

S Supporting Information *

Detailed information about sampling locations, removal rate of anti-influenza drugs in STPs, time-dependent dynamics of antiinfluenza drugs in effluents from STP effluents, ratio of OS to OC in sewage and river water sampling, and age distribution of flu patients during the 2011 influenza season in Kyoto Prefecture with mean amount of Tamiflu taken per patient per day. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-77-527-6222; fax: +81-77-527-9869; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

We thank all of the staff at the STPs for sampling the waters. We acknowledge Japan Science and Technology Agency (JST), the Japan Society for the Promotion of Science, the Ministry of Land, Infrastructure, Transport, and Tourism of Japan, and the Ministry of Education, Culture, Sport, and Science and Technology of Japan for funding in the form of research and scholarships.

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