DSpace at VNU: Ubiquitous occurrence of sulfonamides in tropical Asian waters

Science of the Total Environment 452–453 (2013) 108–115 Contents lists available at SciVerse ScienceDirect Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv Ubiquitous occurrence of sulfonamides in tropical Asian waters Akiko Shimizu a, Hideshige Takada a,⁎, Tatsuya Koike a, Ayako Takeshita a, Mahua Saha a, Rinawati a, Norihide Nakada a, Ayako Murata a, Tokuma Suzuki a, Satoru Suzuki b, Nguyen H Chiem c, Bui Cach Tuyen d, Pham Hung Viet e, Maria Auxilia Siringan f, Charita Kwan f, Mohamad P Zakaria g, Alissara Reungsang h a Laboratory of Organic Geochemistry, Tokyo University of Agriculture and Technology, Fuchu, Tokyo, 183-8509, Japan Marine Molecular Ecology (MME Lab), Center for Marine Environmental Studies (CMES), Ehime University, Matsuyama, 790-8577, Japan College of Agriculture, Can Tho University, Vietnam d Nong Lam University, Ho Chi Minh City, Vietnam e Research Center for Environmental Technology and Sustainable Development, Hanoi University of Science, Hanoi, Vietnam f Natural Sciences Research Institute, University of the Philippines, 1101 Diliman, Quezon City, Philippines g Department of Environmental Sciences, Faculty of Environmental Studies, Universiti Putra Malaysia, 43400 UPM, Serdang, Selangor Darul Ehsan, Malaysia h Department of Biotechnology, Khon Kaen University, Khon Kaen, Thailand b c H I G H L I G H T S ► ► ► ► Sulfonamides, especially sulfamethoxazole, was dominant in sewage in tropical Asia Sulfamethazine and oxytetracycline were dominant in livestock and aquaculture waste ~ 10% of sulfamethoxazole in Mekong River was derived from pig-farm wastewater 12 tons/year of sulfamethoxazole is supplied from Mekong River to South China Sea a r t i c l e i n f o Article history: Received 20 December 2012 Received in revised form February 2013 Accepted February 2013 Available online 15 March 2013 Keywords: Antibiotics Sulfamethoxazole Sulfamethazine Oxytetracycline Mekong Delta Veterinary antibiotics a b s t r a c t Seven sulfonamides, trimethoprim, five macrolides, lincomycin and three tetracyclines were measured in 150 water samples of sewage, livestock and aquaculture wastewater, and river and coastal waters, in five tropical Asian countries The sum of the concentrations of the target antibiotics in sewage and heavily sewage-impacted waters were at sub- to low-ppb levels The most abundant antibiotic was sulfamethoxazole (SMX), followed by lincomycin and sulfathiazole The average concentration of SMX in sewage or heavily sewage-impacted waters was 1720 ng/L in Vietnam (Hanoi, Ho Chi Minh, Can Tho; n=15), 802 ng/L in the Philippines (Manila; n=4), 538 ng/L in India (Kolkata; n=4), 282 ng/L in Indonesia (Jakarta; n=10), and 76 ng/L in Malaysia (Kuala Lumpur; n=6) These concentrations were higher than those in Japan, China, Europe, the US and Canada A predominance of sulfonamides, especially SMX, is notable in these tropical countries The higher average concentrations, and the predominance of SMX, can be ascribed to the lower cost of the antibiotics Both the concentration and composition of antibiotics in livestock and aquaculture wastewater varied widely In many cases, sulfamethazine (SMT), oxytetracycline (OTC), lincomycin, and SMX were predominant in livestock and aquaculture wastewater Both human and animal antibiotics were widely distributed in the respective receiving waters (i.e., the Mekong River and Manila Bay) SMT/SMX ratios indicate a significant contribution from livestock wastewater to the Mekong River and nearby canals, with an estimated ~10% of river water SMX derived from such wastewater Mass flow calculations estimate that 12 tons of SMX is discharged annually from the Mekong River into the South China Sea Riverine inputs of antibiotics may significantly increase the concentration of such antibiotics in the coastal waters © 2013 Elsevier B.V All rights reserved Introduction Human and veterinary antibiotics have been widely detected in municipal and agricultural wastewater and receiving waters (Kümmerer, 2009a) Antibiotics are biologically active, and their ecological impact ⁎ Corresponding author Tel.: +81 42 367 5825; fax: +81 42 360 8264 E-mail address: shige@cc.tuat.ac.jp (H Takada) 0048-9697/$ – see front matter © 2013 Elsevier B.V All rights reserved http://dx.doi.org/10.1016/j.scitotenv.2013.02.027 has been a source of concern Most notably, the emergence of antibiotics resistance is of great concern (Kümmerer, 2009b; Hoa et al., 2008) To assess the ecological impact of antibiotics in aquatic environments, it is essential to understand the types of antibiotics and their concentrations in the respective source (i.e., wastewater) and receiving water There have been reports on environmental antibiotics in North America, Europe and East Asia (e.g., Hirsch et al., 1999; Miao et al., 2004; Göbel et al., 2005; Xu et al., 2007; Watkinson et al., 2007; Spongberg and Witter, A Shimizu et al / Science of the Total Environment 452–453 (2013) 108–115 2008; Lin et al., 2008; Li et al., 2009; Sim et al., 2011; García-Galán et al., 2011; Murata et al., 2011; Segura et al., 2009 and references therein), but these regions are located in cold and temperate climate zones, and very limited information is available on antibiotics in tropical, specifically South and Southeast Asian, waters (Managaki et al., 2007) Infectious disease is more pervasive, and a higher percentage of people suffer from such disease, in tropical Asia than in North America, Europe or East Asia According to WHO data (WHO, 2012), infectious and parasitic diseases account for 19% of total deaths in Southeast Asia, and only 3% in Europe and 5% in Americas This Southeast Asian figure is probably due, in part, to the regional climate conditions (hot and wet), which are conducive to the incubation of vector microorganisms, and to insufficient sewer and water supply systems The higher levels of infectious disease may lead to increased usage of antibiotics, and this, in turn, to higher concentrations of antibiotics in the tropical Asian waters Due to economic factors, inexpensive antibiotics are frequently used in tropical Asian countries and could be detected widely in tropical Asian waters (Dang et al., 2011; Hoa et al., 2011; Takasu et al., 2011) More frequent exposure of bacteria to these antibiotics may develop and select antibiotic resistant bacteria to these antibiotics in the environment even at low concentrations (Knapp et al., 2008; Gullberg et al., 2011) There have, in fact, been several reports examining antibiotic-resistant genes in the tropical Asian aquatic environment (Agersø and Petersen, 2007; Hoa et al., 2008) In addition to human medicine, large amounts of veterinary antibiotics are assumed to be used in the region, due to intensive husbandry and aquaculture activities For example, integrated agricultural operations, such as Vietnam's common ‘vegetable, aquaculture, caged-animal’ (VAC) system, may present an increased risk of human exposure to antibiotics and antibiotic-resistant bacteria/genes (Suzuki and Hoa, 2012) Generally, wastewater treatment, though not totally effective by itself, reduces the environmental burden of antibiotics (Nakada et al., 2007) However, treatment of human waste (sewage) and animal waste is not, at present, adequately implemented in tropical Asian countries, as has been confirmed by the monitoring of molecular markers (Isobe et al., 2002, 2004) Furthermore, climate conditions (particularly, frequent heavy rain) may facilitate the transport of antibiotics to rivers and coastal zones, due to resulting overflows of sewage treatment systems (sewers, wastewater storage ponds, lagoons) and VAC systems, increased agricultural surface runoff, and rapid flushing through streams In light of the foregoing, higher concentrations of antibiotics, especially inexpensive varieties, are a concern in tropical Asian countries However, thus far, only very limited information (Managaki et al., 2007) has been available on the types, abundance, distribution and mass flow of antibiotics in tropical Asian waters In the present study, we measured seven sulfonamides, trimethoprim, five macrolides, lincomycin and three tetracyclines in sewage, canals heavily-impacted by sewage, wastewater from livestock farms and aquacultures, and rivers and coastal waters, in Vietnam, the Philippines, Indonesia, Malaysia and India These antibiotics are the most commonly used worldwide Materials and methods 2.1 Study areas and samples The sample details, including coordinates, dates, pH and electric conductivity, are listed in Tables A1, A2 and A3 Water samples were collected from urban drainage, canals and heavily sewage-impacted rivers in Vietnam (Hanoi, Ho Chi Minh City, Can Tho), the Philippines (Manila), Indonesia (Jakarta), India (Kolkata) and Malaysia (Kuala Lumpur), from 2006 to 2010 (Fig 1) In the study areas, sewage treatment systems not serve all residents and untreated sewage are directly discharged to canals and rivers The percentage of the population served by sewage treatment ranged from 0% in Can Tho to 75% in Kuala Lumpur as indicated in Table A4 Livestock wastewater (pig-, cow-, 109 chicken-farm wastewater and aquaculture wastewater) was collected in Vietnam (Hanoi, Ho Chi Minh City, Can Tho) and Thailand (Kohn Kaen) In Can Tho, water samples were collected from both rural and urban canals River water samples were collected from the Mekong River, to investigate horizontal distribution, and diurnal and seasonal change, in antibiotic levels In Manila, water samples were collected from Manila Bay, Laguna Bay and the Pasig River 2.2 Analytical procedure HPLC-grade solvent (water, methanol (MeOH), acetonitrile), ethylenediaminetetraacetic acid (EDTA; >99.5% purity), and formic acid (>99.5%) were supplied by Wako Pure Chemicals (Osaka, Japan) Sources, purity and acronyms of antibiotics standards, icluding isotopically-labeled antibitotics standards, are described in Table The samples were collected with a stainless steel bucket, stored in 1-L amber plastic bottles, transported cool to the laboratory, and filtered through pre-baked glass fiber filters (GF/F, Whatman) Antibiotics were analyzed according to Ye et al (2006) with slight modifications Antibiotics in the filtrates were extracted with 6-mL solid-phase extraction (SPE) cartridges (200 mg Oasis HLB resin, Waters) The cartridges were preconditioned with mL methanol, mL methanol containing 0.1% (v/v) formic acid, and × 1.5 mL water To adjust pH and prevent the chelation of tetracyclines with metal cations, EDTA was added as a solid and mixed, such that the EDTA concentration in the filtrate was 0.1% Aliquots of the filtrate, with volumes ranging from 50 mL for sewage and animal wastes to 200 mL for river water as listed in Tables A5, and 7, were passed through the conditioned SPE cartridges After the extraction, water in SPE cartridges was removed by passing air for 30 s and the SPE cartridges containing the antibiotics were wrapped with aluminum foil and placed in a Ziploc bag The cartridges were packed in dry ice during transport to the laboratory in Japan, then stored in a freezer at −30 °C until the time of analysis Just before analysis, each SPE cartridge was thawed and placed on a vacuum manifold The cartridge was washed with × mL of water and dried in an air flow for The analyte was then eluted with × mL methanol containing 0.1% (v/v) formic acid The eluent was combined and spiked with an appropriate volume (50–200 μL) of an internal standard mixture consisting of sulfamethoxazole-d4, clarithromycin-d3, roxithromycin-d9, and oxytetracycline- 13C1, -d3 (500 ppb each, in methanol) The eluent was then concentrated to around 0.5 mL in a rotary evaporator, and transferred to a mL amber vial The eluent in the vial was evaporated to complete dryness under a nitrogen stream at 80 °C, and dissolved in an appropriate volume (0.5 mL–40 mL) of H2O/acetonitrile (94:6 v/v) containing 0.1% formic acid The volume of the final solution is also listed in Tables A5, 6, and to allow the calculation of the preconcentration factor, which ranged from to 400 A 20-μL aliquot was injected into a liquid chromatograph (Agilent series 1100, Tokyo, Japan) equipped with a tandem mass spectrometer (TSQ Quantum 7000, Thermo Finnigan, Japan) The antibiotics quantified by mass spectrometer were separated in an Xterra MS C18 (2.1 mm i.d.× 50 mm; particle size: 2.5 μm; Waters) with a guard column (Xterra MS C18; 2.1 mm i.d.×20 mm; particle size: 3.5 μm; Waters), using a binary gradient system (solvent A: 0.1% formic acid in H2O; solvent B: acetonitrile), at a flow rate of 0.2 mL/min The run started at 5% B for min, followed by a 11-min linear gradient to 95% B, after which the initial conditions were reestablished and the column was equilibrated for 17 Analytes were quantified in selected reaction monitoring (SRM) mode with positive electrospray ionization (ESI) in positive mode The operating conditions of the mass spectrometer are listed in Table A8 The m/z values of the precursur ion (Q1) and two monitored product ions (Q3) are listed in Table A9 To identify antibiotics, we compared the retention times and the area ratios of the two product ions in each sample with the average retention time and peak ratios of standards in all measurements The criteria difference between samples and the standard was within 110 A Shimizu et al / Science of the Total Environment 452–453 (2013) 108–115 (a) (b) (c) Fig (a) Study area Red circles: cities investigated, (b) Sampling locations in Can Tho and the Mekong River VMR1–4 and VMR 5–7: cross sections for investigation of horizontal distribution of antibiotics in the river VMR21 and VMR23: locations for time-series observations, (c) Sampling locations in Manila Bay, the Pasig River and Laguna Lake 0.3 for the retention time and 20% for the area ratio of the two product ions External calibration curves (area of individual components as a function of their concentrations) were used for quantifiction Calibration lines of the individual antibiotics, with concentration levels (1, 3, 5, 10, 20, 30, 40, and 50 μg/L), were used on a routine basis The linearity of the calibration curve in this range was confirmed (R2 > 0.99) For several compounds with high responses (SMT, SDX, CLA, ROX, TRI, LIN), linearlity (R2 >0.99) of calibration line down to 0.1 μg/L was confirmed by using series of standards including 0.1 μg/L Final concentrations of most of the samples in the vials were within the range of the calibration lines When the final concentrations were lower than the lowest standard concentration (1 μg/L), the concentration in the sample was calculated by interpolation from the calibration lines between μg/L and the origin The concentrations of the target antibiotics were corrected against the recovery of internal standards, as listed in Table A9 A Shimizu et al / Science of the Total Environment 452–453 (2013) 108–115 Table Target antibiotics and their sources Compound name Acronym Category Supplier Purity (%) Sulfapyridine Sulfamethoxazole SPY SMX Sulfonamide Sulfonamide Sulfathiazole STZ Sulfonamide Sulfamerazine SMR Sulfonamide SMZ Sulfonamide SMT Sulfonamide Sulfadimethoxine SDX Sulfonamide Trimethoprim TRI 2,4-Diaminopyrimidine Azithromycin AZI Macrolide Erythromycin ERYb Macrolide Clarithromycin CLA Macrolide Roxithromycin Tylosin ROX TYL Macrolide Macrolide Lincomycin LIN Lincosamide Tetracycline Doxycycline TC DOX Cycline Cycline Oxytetracycline OTC Cycline Sulfamethoxazole-d4 d-SMX Sulfonamide, labeled Clarithromycin-d3 d-CLA Macrolide, labeled Roxithromycin-d9 d-ROX Macrolide, labeled Oxytetracycline-13C, d3 d-OTC Cycline, labeled Sulfamethizole Sulfamethazine a b a Sigma Wako pure chemicals Riedel-de Haen Wako pure chemicals Riedel-de Haen Wako pure chemicals Wako pure chemicals Wako pure chemicals LKT laboratories, Inc Riedel-de Haen Wako pure chemicals Sigma Riedel-de Haen Riedel-de Haen Sigma ICN Biomedicals Inc Wako pure chemicals Hayashi pure chemicals Hayashi pure chemicals Hayashi pure chemicals Hayashi pure chemicals >99.0 >99.0 99.9 >99.0 99.9 >99.0 >99.0 >99.0 >98 99.9 >95 90.0 91.0 100 97.4 >83.9 111 reproducibility and of recovery, the internal standards and native standards (only for recovery test) were spiked before solid-phase extraction and the SPE cartridges were not frozen However, for the overseas samples, solid phase extraction was done on-site, without spiking the internal standards, and the SPE cartridges were frozen and brought back to Japan, then stored in a freezer until analysis Immediately before the analysis, each SPE cartridge was thawed and eluted with MeOH, and then the internal standards were spiked The estimated error associated with the overseas procedure appears in Table STP effluents were analyzed both with normal laboratory procedure (i.e., internal standard spiking before SPE, no freezing of SPE cartridge) and with the overseas sample procedure (i.e., internal standard spiking after SPE, freezing of SPE cartridge) Difference between the two procedures was less than 15% for all the detected antibiotics, except for TRI (19%) and ROX (18%) In addition, in order to measure the error for the antibiotics not present in the STP effluents, we spiked native antibiotics standards to the effluent sample and compared the results of analysis using the two procedures described above An error increase of 25% was observed for AZI and 24% error decrease was observed for OTC in the case of the overseas procedure, in comparison with the normal laboratory procedure Based on these examinations, reported values in the present study (i.e., internal standard spiking after SPE, freezing of SPE cartridge) should be considered to have 25% of inaccuracy at most The inaccuracy was derived from integration of all the factors associated with oversea procedure, e.g., compound losses during extraction, shipment, freezing and increases due to cleavage of conjugates during thawing Results and discussion 3.1 Antibiotics in sewage and livestock wastewater 99.0 >97.2 n.a n.a n.a Sulfadimidine Acronym “ERY” in the present paper means dehydrated erythromycin 2.3 Analytical performance Solvent blanks (H2O/acetonitrile (94:6 v/v) containing 0.1% formic acid) were used to measure noise levels for individual antibiotics and calculate the limit of detection (LOD) The LOD was determined as the concentration equivalent to a signal-to-noise (S/N) ratio of for each set of sample analysis (normally samples per set) Procedural blanks (i.e extracted blanks) were run for each set of sample analysis and were used to calculate the limit of quantification (LOQ) The LOQ was defined as 10 times the procedural blank value When no peak occurred for a certain antibiotic, the S/N = 10 was used as LOQ In addition, when the LOQ was below the lowest concentration level of the calibration curve (0.1 μg/L for SPY, SMT, SDX, CLA, ROX, TRI, LIN or μg/L for the other compounds), the concentration calculated based on the lowest standard concentration was considered as LOQ LOD and LOQ were normally 0.08 ng/L–0.8 ng/L and 0.2 ng/L–2 ng/L, respectively, as listed in Tables A10 and A11, respectively Reproducibility was determined by triplicate analysis of effluent from a sewage treatment plant (STP) Relative standard deviations of concentrations of the target compounds were ≤12% (Table A12) Recovery throughout the analytical procedure was checked by replicate analysis (n= 4) of the STP effluent spiked with standard mixtures Recoveries of the antibiotics were ≥79% (Table A13) For these tests of The sum of the concentrations of the target antibiotics in sewage and sewage-impacted waters in tropical Asian countries were at sub- to low-ppb levels (Fig 2, Table A5) The average total concentration was 3220 ng/L for Vietnam, 1576 ng/L for the Philippines, and 607 ng/L for Indonesia In the case of the Indian and Malaysian samples, tetracyclines were not analyzed, and therefore these samples were excluded from discussion of the total concentrations and profiles of the antibiotics Compositions of antibiotics were considerably constant within sewage and sewage-impacted rivers for individual countries Among the target antibiotics, sulfonamides, especially sulfamethoxazole (SMX), were predominant (Fig 3, Fig A1), in contrast to the antibiotics composition in Japan where macrolides were predominant The ratio of sulfonamides to macrolides (average ± standard deviation) was 3.6 ± 3.6 for Vietnam, 1.9 ± 0.4 for the Philippines, 6.2± 3.3 for Indonesia, 3.4 ±1.7 for India, and 0.7 ± 0.2 for Malaysia, compared to 0.3± 0.03 for sewage in Tokyo (Morimoto et al., 2011) The predominance of sulfonamides, especially SMX, is notable in tropical Asian countries Both sulfonamides and macrolides are used to treat the same infections (e.g., Staphylococcus, Enterococcus and Lactcoccus diseases) The reason behind the dominant usage of sulfonamides could be their lower price The secondmost abundant antibiotic in Vietnam and Indonesia was lincomycin, while erythromycin-H2O was predominant in the Philippines (Fig 3) The average concentration of SMX in sewage or heavily sewageimpacted waters was 1720 ng/L in Vietnam (Hanoi, Ho Chi Minh, Can Tho; n = 15), 802 ng/L in the Philippines (Manila; n = 4), 538 ng/L in India (Kolkata; n = 4), 282 ng/L in Indonesia (Jakarta; n = 10), and 76 ng/L in Malaysia (Kuala Lumpur; n = 6) (Fig 4) SMX concentrations in most of the tropical Asian countries (except for Malaysia and Indonesia) were higher than those in Japan (88 ng/L; Morimoto et al., 2011), China (10–370 ng/L; Li et al., 2009; Xu et al., 2007), Taiwan (226 ng/L; Lin et al., 2008), Korea (295 ng/L; Sim et al., 2011), Australia (360 ng/L; Watkinson et al., 2007), Europe (Spain: 12–650 ng/L (García-Galán et al., 2010, 2011); Switzerland: 430 ng/L (Göbel et al., 2005); Germany: 400 ng/L (Hirsch et al., 1999), the US (135–261 ng/L; Spongberg and Witter, 2008), and Canada (243 ng/L; 112 A Shimizu et al / Science of the Total Environment 452–453 (2013) 108–115 Table Accuracy of antibiotics concentrations by using oversea procedure Internal standard spiking Native standards spiking Freeze SPY SMX STZ SMR SMZ SMT SDX TRI AZI ERY CLA ROX TYL LIN TC DOX OTC Amounts in the sample (ng/L) Oversea/Labo Amounts in the sample (ng/vial) Oversea/Labo Laboratory procedure Oversea procedure (%) Laboratory procedure Oversea procedure (%) Before SPE Non Non 278 91 0 0 65 439 245 455 94 Trace Trace Trace 90 After SPE Non Freeze 268 80 0 0 53 384 230 392 77 Trace Trace Trace 96 96 88 n.a n.a n.a n.a n.a 81 87 94 86 82 n.a n.a n.a n.a 107 Before SPE Before SPE Non 166 186 139 138 139 177 176 194 252 234 187 188 180 271 280 221 260 After SPE Before SPE Freeze 185 195 153 154 159 194 170 230 316 248 201 186 185 275 248 184 198 111 105 110 112 114 109 96 119 125 106 108 99 103 101 89 83 76 Miao et al., 2004), as shown in Fig The higher concentrations of SMX in tropical Asian countries could be partially due to lower access to sewage treatment systems as indicated in Table A4 Substantial removal of SMX through sewage treatment (primary and secondary) has been reported (~60%: García-Galán et al., 2011; ~30%: Morimoto et al., 2011) However, more likely the reason for the higher concentrations, and the predominance of SMX is the high amounts of this antibiotic used in those countries Heavier usage could be ascribed to the lower cost of the antibiotics Sulfamethoxazole is a relatively inexpensive antibiotic, in comparison with macrolides Table A14 shows the cost of antibiotics sold in Vietnam The cost per tablet of sulfamethoxazole is one order of magnitude lower than that of macrolides (clarithromycin and roxithromycin) It is thought that people in low-income countries typically use low-cost medicine such as SMX The concentration and composition of livestock and aquaculture wastewater varied widely (Fig and Table A6) In many cases, sulfamethazine (SMT), oxytetracycline (OTC), lincomycin and SMX were predominant among the target antibiotics (Fig and Table A6) However, antibiotics composition varied widely even among livestock wastewater from the same area For example, among pig-farm effluent samples (AW1–AW4), collected in Can Tho on the same day, there was variance in the most abundant antibiotic: SMX for AW1, SMT for AW2, LIN for AW3, and OTC for AW4; though SMT was detected in all the effluent samples (Fig A2) This variation in antibiotic composition can be explained by the fact that the application of antibiotics depends on specific animals and specific diseases, and sampling was done at the outfalls of individual farms or aquacultures Though the antibiotic composition varied widely in the livestock and aquaculture wastewater, Fig Concentrations of antibiotics in sewage, urban drainage, and livestock and aquaculture wastewater, and river, lake and coastal waters, in tropical Asian countries Total antibiotics: sum of all the target antibiotics listed in Table 1, except for India and Malaysia (where only sulfonamides and macrolides were measured) A Shimizu et al / Science of the Total Environment 452–453 (2013) 108–115 113 Fig Average composition of antibiotics in sewage, urban drainage, and livestock and aquaculture wastewater, and river, lake and coastal waters, in tropical Asian countries and Japan SMT can be regarded as livestock wastewater-derived antibiotics, because they were consistently detected in the livestock wastewater samples Fig Concentrations of sulfamethoxazole in sewage, urban drainage, and heavily sewage-affected rivers, in tropical Asian countries, in comparison with those reported for other countries a) Morimoto et al (2011); b) Lin et al (2008); c) Li et al (2009), Xu et al (2007); d) Sim et al (2011); e) Watkinson et al (2007); f) Hirsch et al (1999); g) Göbel et al (2005); h) García-Galán et al (2010); i) García-Galán et al (2011); j) Miao et al (2004); k) Spongberg and Witter (2008) 3.2 Contribution of human and veterinary antibiotics to natural waters Both human and animal antibiotics were widely distributed in the respective receiving waters (i.e., the Mekong Delta, and Manila Bay and vicinity) (Fig 3, Table A7) Antibiotic compositions in the Mekong River were basically similar to those in sewage, suggesting that sewage was the major contributor of antibiotics to the river (Fig 3) However, the proportion of SMT, which were abundant in livestock wastewater, was significantly higher in the Mekong River (20.7% of total antibiotics) than in sewage (2.3%), suggesting a minor but significant contribution from livestock and aquaculture wastewater in the case of the Mekong River A similarly significant contribution from livestock wastes was apparent in the riverine and coastal waters in Manila, Philippines The percentage SMT of total antibiotics in the Pasig River (including Laguna Lake) and Manila Bay was 23.8%± 9.3% and 21.2%± 9.3%, respectively, compared to 1.9% ± 1.9% in the case of sewage (i.e., canal water) from the same area To enable quantitative discussion of the contribution from livestock wastes, the SMT/SMX ratio was calculated as an index to estimate the relative contribution of livestock wastes according to Managaki et al (2007) The SMT/SMX ratio in sewage in Vietnam ranged from 0.009 to 0.343, with a geometric mean of 0.043 (Fig 5, Table A5) In contrast, the SMT/SMX ratio was much higher (up to 600 times greater) in many instances of livestock waste in Vietnam The geometric mean of SMT/SMX ratio in pig-farm wastewater in Vietnam was 2.1 (Table A6) The SMT/SMX ratio in the Mekong River ranged from 0.17 to 0.97, with a geometric mean of 0.34 (Table A7), and these were higher than in sewage and lower than in livestock waste, indicating that both sewage and livestock waste contribute to the antibiotics in the river water of the Mekong This trend is consistent with the SMT/SMX ratios in urban and rural canals; the former had similarly lower ratios (~0.1), and the latter, higher values (~0.4) Rural canals in Vietnam receive both sewage and livestock waste Using the mean SMT/SMX ratios in sewage (0.043), livestock waste (pig-farm wastewater: 2.1), and Mekong River water (0.34), 114 A Shimizu et al / Science of the Total Environment 452–453 (2013) 108–115 Fig Diurnal change in antibiotics concentrations in the Mekong River (VMR 21 for the wet season, VMR 23 for the dry season) Fig Ratio of sulfamethazine to sulfamethoxazole (SMT/SMX ratio) in sewage, pig-farm wastewater, canals and the Mekong River the contribution from livestock waste to SMX levels in the Mekong River was roughly estimated at 14% The detailed calculation procedure is described in Table A15 This relatively large contribution of livestockderived antibiotics stands in contrast to the negligible contribution of livestock wastewater to Japanese rivers (Murata et al., 2011) Furthermore, taking the geometric mean concentration of SMX in sewage (988 ng/L) and pig-farm wastewater (148 ng/L) into the calculation, the total volume of livestock wastewater is estimated to be comparable (52:48) to the volume of sewage discharged to the Mekong River The large contribution of livestock wastewater effluents, comparable to sewage, is probably owing to the combination of large numbers of livestock in the region, and insufficient treatment of livestock wastes For example, in the catchment of the Mekong Delta, the ratio of the number of pigs (3.7×10 6) to people (17×106) is 0.2, whereas for the whole of Japan it is 0.08 (10×106 pigs to 120×106 people) In addition, the frequent and heavy rainfall, common to the tropical area, facilitates overflow of livestock wastes from the retention ponds, including VAC wastewater and agricultural runoff containing animal wastes However, it should be stressed that variations in the SMT/SMX ratio in the source materials, especially in livestock wastes, would cause inaccuracies in the estimation To increase the accuracy of assessments based on this approach, more numerous and more representative samples of livestock wastewater are necessary 3.3 Mass flow of antibiotics in the Mekong river Horizontal variation of SMX concentrations in the Mekong River was examined, in the Can Tho area, by establishing two cross sections of the river, above and below Can Tho City (Fig 1-b) SMX concentrations across both cross sections were fairly constant, with a roughly 10% variation, as shown in Fig A3 These data suggest that the antibiotics were horizontally well mixed Moreover, our time-series observation at locations on the Mekong River (VMR21 and VMR23) demonstrated no great diurnal variation in SMX concentrations in the Mekong River (Fig 6) SMX concentrations were higher in the dry season (December, 2010) than in the wet season (September, 2008 and 2009) by a factor of at the most This may be attributable to dilution by rain water in the wet season These observations indicate that temporal and horizontal variation in SMX concentrations in Mekong River falls within a factor of Mass flow of SMX is calculated at 12 tons per year, based on the average concentrations of SMX in the river water (25 ng/L) and annual discharge of river water (475 km3; Mekong River Commission, 2010) Taking into account seasonal variations, to 24 tons of SMX is estimated to be discharged annually into the South China Sea (SCS), potentially representing a significant contribution to SMX levels in the SCS Assuming that 12 tons of SMX is mixed with the surface layer (roughly 200 m) of the SCS (350 × 10 km2), and no removal occurs, there would be an estimated 0.2 ng/L increase in SMX in the seawater of the SCS If this situation continued for the coming decade, the overall increase in SCS seawater SMX concentration would be ng/L SMX is water-soluble, and thus its removal from the water column, through adsorption onto particles and their settling, would not be significant In addition, SMX is resistant to microbial degradation, with half the total amount of SMX remaining after activated sludge treatment (Nakada et al., 2007) Photodegradation, especially under the strong sunlight typical of tropical environments, would decrease the concentrations of SMX Otherwise, gradual but significant increases in the concentration of antibiotics in the region's seawater may be occurring The effects, on the coastal ecosystem, of low but significant levels of antibiotics in seawater, should be assessed in future studies Conclusion A predominance of sulfonamides, especially sulfamethoxazole (SMX), was observed in sewage and heavily sewage-impacted waters of tropical Asian countries, probably due to the lower cost of sulfonamide antibiotics Sulfamethazine (SMT) was predominant in livestock animal wastewater, though the respective antibiotic compositions were more variable than those in sewage Based on SMT/SMX ratios, it was estimated that roughly 10% of the amount of SMX, and ~50% of the total wastewater volume, of Mekong Delta river water, is derived from livestock wastewater The SMX concentration in the Mekong River showed little horizontal or diurnal variation, but significant seasonal variation (4 times higher in the dry season than in the wet season) Approximately 12 tons of SMX was estimated to be delivered annually to the South China Sea from the Mekong River Conflict of interest The authors declare that there are no conflict of interest Acknowledgments Students and staffs in LOG and foreign counter-part laboratories provided welcome assistance with the fieldwork We thank Dr Pedro A Segura for the comments on the manuscript This research was financially supported by Grant-in-Aid studies from the Ministry of Education and Culture of Japan (Project Nos 22254001, 19405004, and 20405044) Appendix A Supplementary data Supplementary data to this article can be found online at http:// 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ROX, TRI, LIN), linearlity (R2 >0.99) of calibration line down to 0.1 μg/L was confirmed by using series of standards including 0.1 μg/L Final concentrations of most of the samples in the vials... be attributable to dilution by rain water in the wet season These observations indicate that temporal and horizontal variation in SMX concentrations in Mekong River falls within a factor of Mass... Erythromycin ERYb Macrolide Clarithromycin CLA Macrolide Roxithromycin Tylosin ROX TYL Macrolide Macrolide Lincomycin LIN Lincosamide Tetracycline Doxycycline TC DOX Cycline Cycline Oxytetracycline