(This is a sample cover image for this issue. The actual cover is not yet available at this time.

This article appeared in a journal published by Elsevier. The attached

copy is furnished to the author for internal non-commercial research
and education use, including for instruction at the authors institution
and sharing with colleagues.
Other uses, including reproduction and distribution, or selling or
licensing copies, or posting to personal, institutional or third party
websites are prohibited.
In most cases authors are permitted to post their version of the
article (e.g. in Word or Tex form) to their personal website or
institutional repository. Authors requiring further information
regarding Elseviers archiving and manuscript policies are
encouraged to visit:
http://www.elsevier.com/copyright

Author's personal copy

Science of the Total Environment 452-453 (2013) 108115

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 9 February 2013
Accepted 9 February 2013
Available online xxxx
Keywords:
Antibiotics
Sulfamethoxazole
Sulfamethazine
Oxytetracycline
Mekong Delta
Veterinary antibiotics

a b s t r a c t
Seven sulfonamides, trimethoprim, ve macrolides, lincomycin and three tetracyclines were measured in 150
water samples of sewage, livestock and aquaculture wastewater, and river and coastal waters, in ve 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 signicant contribution from livestock wastewater to the Mekong River and nearby canals, with an estimated ~10% of river water SMX derived from such
wastewater. Mass ow calculations estimate that 12 tons of SMX is discharged annually from the Mekong River
into the South China Sea. Riverine inputs of antibiotics may signicantly increase the concentration of such antibiotics in the coastal waters.
2013 Elsevier B.V. All rights reserved.

1. Introduction
Human and veterinary antibiotics have been widely detected in municipal and agricultural wastewater and receiving waters (Kmmerer,
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 (Kmmerer, 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; Gbel et
al., 2005; Xu et al., 2007; Watkinson et al., 2007; Spongberg and Witter,

Author's personal copy

A. Shimizu et al. / Science of the Total Environment 452-453 (2013) 108115

2008; Lin et al., 2008; Li et al., 2009; Sim et al., 2011; Garca-Galn 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, specically 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
gure is probably due, in part, to the regional climate conditions (hot
and wet), which are conducive to the incubation of vector microorganisms, and to insufcient 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
(Agers 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 conrmed 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 overows of sewage treatment systems (sewers,
wastewater storage ponds, lagoons) and VAC systems, increased agricultural surface runoff, and rapid ushing 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 ow of antibiotics in tropical Asian waters.
In the present study, we measured seven sulfonamides, trimethoprim, ve 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.
2. 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 do 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 1.
The samples were collected with a stainless steel bucket, stored in
1-L amber plastic bottles, transported cool to the laboratory, and ltered
through pre-baked glass ber lters (GF/F, Whatman). Antibiotics were
analyzed according to Ye et al. (2006) with slight modications. Antibiotics in the ltrates were extracted with 6-mL solid-phase extraction
(SPE) cartridges (200 mg Oasis HLB resin, Waters). The cartridges
were preconditioned with 6 mL methanol, 3 mL methanol containing
0.1% (v/v) formic acid, and 2 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 ltrate was
0.1%. Aliquots of the ltrate, with volumes ranging from 50 mL for sewage and animal wastes to 200 mL for river water as listed in Tables A5, 6
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 2 6 mL of water
and dried in an air ow for 5 min. The analyte was then eluted with
4 2 mL methanol containing 0.1% (v/v) formic acid. The eluent was
combined and spiked with an appropriate volume (50200 L) 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 4 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 mL40 mL) of H2O/acetonitrile (94:6 v/v) containing 0.1%
formic acid. The volume of the nal solution is also listed in Tables
A5, 6, and 7 to allow the calculation of the preconcentration factor,
which ranged from 5 to 400. A 20-L 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 quantied by mass spectrometer were separated
in an Xterra MS C18 (2.1 mm i.d. 50 mm; particle size: 2.5 m;
Waters) with a guard column (Xterra MS C18; 2.1 mm i.d.20 mm; particle size: 3.5 m; Waters), using a binary gradient system (solvent A:
0.1% formic acid in H2O; solvent B: acetonitrile), at a ow rate of
0.2 mL/min. The run started at 5% B for 5 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 min. Analytes were quantied
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

Author's personal copy

110

A. Shimizu et al. / Science of the Total Environment 452-453 (2013) 108115

(a)

(b)

(c)

Fig. 1. (a) Study area. Red circles: cities investigated, (b) Sampling locations in Can Tho and the Mekong River. VMR14 and VMR 57: 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. (For interpretation of the references to color in this gure legend, the reader is referred to the web version of the article.)

0.3 min 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 quantiction.
Calibration lines of the individual antibiotics, with 8 concentration
levels (1, 3, 5, 10, 20, 30, 40, and 50 g/L), were used on a routine
basis. The linearity of the calibration curve in this range was conrmed
(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 g/L was conrmed by using series of standards including 0.1 g/L.
Final concentrations of most of the samples in the vials were within
the range of the calibration lines. When the nal concentrations were
lower than the lowest standard concentration (1 g/L), the concentration in the sample was calculated by interpolation from the calibration
lines between 1 g/L and the origin. The concentrations of the target antibiotics were corrected against the recovery of internal standards, as
listed in Table A9.

Author's personal copy

A. Shimizu et al. / Science of the Total Environment 452-453 (2013) 108115
Table 1
Target antibiotics and their sources.
Compound name

Acronym

Category

Supplier

Purity
(%)

Sulfapyridine
Sulfamethoxazole

SPY
SMX

Sulfonamide
Sulfonamide

Sulfathiazole

STZ

Sulfonamide

Sulfamerazine

SMR

Sulfonamide

Sulfamethizole

SMZ

Sulfonamide

Sulfamethazinea

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

a
b

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 2. STP efuents 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 efuents, we spiked native antibiotics standards to
the efuent 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.
3. 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 3 for each set of sample analysis (normally 8 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 quantication (LOQ). The LOQ was dened 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 g/L for SPY, SMT,
SDX, CLA, ROX, TRI, LIN or 1 g/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/L0.8 ng/L
and 0.2 ng/L2 ng/L, respectively, as listed in Tables A10 and A11,
respectively.
Reproducibility was determined by triplicate analysis of efuent
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 efuent 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 proles 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 (10370 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:
12650 ng/L (Garca-Galn et al., 2010, 2011); Switzerland: 430 ng/L
(Gbel et al., 2005); Germany: 400 ng/L (Hirsch et al., 1999), the US
(135261 ng/L; Spongberg and Witter, 2008), and Canada (243 ng/L;

Author's personal copy

112

A. Shimizu et al. / Science of the Total Environment 452-453 (2013) 108115

Table 2
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
0
0
0
65
439
245
455
94
Trace
0
Trace
Trace
90

After SPE
Non
Freeze
268
80
0
0
0
0
0
53
384
230
392
77
Trace
0
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. 4. 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%: Garca-Galn 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. 3 and Table A6). In many cases,
sulfamethazine (SMT), oxytetracycline (OTC), lincomycin and SMX
were predominant among the target antibiotics (Fig. 3 and Table A6).
However, antibiotics composition varied widely even among livestock
wastewater from the same area. For example, among pig-farm efuent
samples (AW1AW4), 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
efuent samples (Fig. A2). This variation in antibiotic composition can
be explained by the fact that the application of antibiotics depends on
specic animals and specic 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. 2. 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).

Author's personal copy

A. Shimizu et al. / Science of the Total Environment 452-453 (2013) 108115

113

Fig. 3. 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. 4. 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) Gbel et al. (2005); h) Garca-Galn et al. (2010); i) Garca-Galn 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 signicantly higher in the Mekong River (20.7% of total antibiotics)
than in sewage (2.3%), suggesting a minor but signicant contribution
from livestock and aquaculture wastewater in the case of the Mekong
River. A similarly signicant 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),

Author's personal copy

114

A. Shimizu et al. / Science of the Total Environment 452-453 (2013) 108115

Fig. 6. Diurnal change in antibiotics concentrations in the Mekong River (VMR 21 for
the wet season, VMR 23 for the dry season).

Fig. 5. 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 efuents, comparable to
sewage, is probably owing to the combination of large numbers of livestock in the region, and insufcient treatment of livestock wastes. For
example, in the catchment of the Mekong Delta, the ratio of the number
of pigs (3.710 6) to people (17106) is 0.2, whereas for the whole of
Japan it is 0.08 (10106 pigs to 120106 people). In addition, the
frequent and heavy rainfall, common to the tropical area, facilitates overow 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 ow 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 4 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 4.
Mass ow 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, 6 to 24 tons of SMX is estimated to be discharged annually into the South China Sea (SCS), potentially
representing a signicant 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 4 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 2 ng/L. SMX is water-soluble,
and thus its removal from the water column, through adsorption onto
particles and their settling, would not be signicant. 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 signicant increases in the concentration of antibiotics in
the region's seawater may be occurring. The effects, on the coastal ecosystem, of low but signicant levels of antibiotics in seawater, should
be assessed in future studies.
4. 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 signicant 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.
Conict of interest
The authors declare that there are no conict of interest.
Acknowledgments
Students and staffs in LOG and foreign counter-part laboratories provided welcome assistance with the eldwork. We thank Dr. Pedro A.
Segura for the comments on the manuscript. This research was nancially 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://
dx.doi.org/10.1016/j.scitotenv.2013.02.027.
References
Agers Y, Petersen A. The tetracycline resistance determinant Tet 39 and the
sulphonamide resistance gene sulII are common among resistant Acinetobacter
spp. isolated from integrated sh farms in Thailand. J Antimicrob Chemother
2007;59:237.

Author's personal copy

A. Shimizu et al. / Science of the Total Environment 452-453 (2013) 108115
Dang STT, Petersen A, Truong DV, Chu HTT, Dalsgaard A. Impact of medicated feed on the
development of antimicrobial resistance in bacteria at integrated pig-sh farms in
Vietnam. Appl Environ Microbiol 2011;77:44948.
Garca-Galn MJ, Daz-Cruz MS, Barcel D. Determination of 19 sulfonamides in
environmental water samples by automated on-line solid-phase extraction-liquid
chromatography-tandem mass spectrometry (SPE-LC-MS/MS). Talanta 2010;81:
35566.
Garca-Galn MJ, Daz-Cruz MS, Barcel D. Occurrence of sulfonamide residues along
the Ebro river basin: removal in wastewater treatment plants and environmental
impact assessment. Environ Int 2011;37:46273.
Gbel A, Thomsen A, McArdell CS, Joss A, Giger W. Occurrence and sorption behavior of
sulfonamides, macrolides, and trimethoprim in activated sludge treatment. Environ
Sci Technol 2005;39:39819.
Gullberg E, Cao S, Berg OG, Ilbck C, Sandegren L, Hughes D, et al. Selection of resistant
bacteria at very low antibiotic concentrations. PLoS Pathog 2011;7:e1002158.
Hirsch R, Ternes T, Haberer K, Kratz K-L. Occurrence of antibiotics in the aquatic
environment. Sci Total Environ 1999;225:10918.
Hoa PTP, Nonaka L, Viet PH, Suzuki S. Detection of the sul1, sul2, and sul3 genes in
sulfonamide-resistant bacteria from wastewater and shrimp ponds of north Vietnam.
Sci Total Environ 2008;405:37784.
Hoa PTP, Managaki S, Nakada N, Takada H, Shimizu A, Anh DH, et al. Antibiotic
contamination and occurrence of antibiotic-resistant bacteria in aquatic environments
of northern Vietnam. Sci Total Environ 2011;409:2894901.
Isobe KO, Tarao M, Zakaria MP, Chiem NH, Minh le Y, Takada H. Quantitative application
of fecal sterols using gas chromatographymass spectrometry to investigate fecal
pollution in tropical waters: western Malaysia and Mekong Delta, Vietnam. Environ
Sci Technol 2002;36:4497507.
Isobe KO, Zakaria MP, Chiem NH, Minh LY, Prudente M, Boonyatumanond R, et al.
Distribution of linear alkylbenzenes (LABs) in riverine and coastal environments
in South and Southeast Asia. Water Res 2004;38:244959.
Knapp CW, Engemann CA, Hanson ML, Keen PL, Hall KJ, Graham DW. Indirect evidence
of transposon-mediated selection of antibiotic resistance genes in aquatic systems
at low-Level oxytetracycline exposures. Environ Sci Technol 2008;42:534853.
Kmmerer K. Antibiotics in the aquatic environment a review part I. Chemosphere
2009a;75:41734.
Kmmerer K. Antibiotics in the aquatic environment a review part II. Chemosphere
2009b;75:43541.
Li B, Zhang T, Xu Z, Fang HH. Rapid analysis of 21 antibiotics of multiple classes in municipal
wastewater using ultra performance liquid chromatography-tandem mass
spectrometry. Anal Chim Acta 2009;645:6472.
Lin AY-C, Yu T-H, Lin C-F. Pharmaceutical contamination in residential, industrial, and
agricultural waste streams: risk to aqueous environments in Taiwan. Chemosphere
2008;74:13141.

115

Managaki S, Murata A, Takada H, Tuyen BC, Chiem NH. Distribution of macrolides,

sulfonamides, and trimethoprim in tropical waters: ubiquitous occurrence of veterinary
antibiotics in the Mekong Delta. Environ Sci Technol 2007;41:800410.
Mekong River Commission. In: MRC, editor. State of the basin report; 2010. [Vientiane,
Laos].
Miao X-S, Bishay F, Chen M, Metcalfe CD. Occurrence of antimicrobials in the nal efuents
of wastewater treatment plants in Canada. Environ Sci Technol 2004;38:353341.
Morimoto T, Muramatsu Y, Takeshita A, Shimizu A, Murakami M, Takada H. Concentrations
of water-soluble organic micropollutants and anthropogenic molecular markers in
sewage inuent and efuent. J Water Waste 2011;53:63546.
Murata A, Takada H, Mutoh K, Hosoda H, Harada A, Nakada N. Nationwide monitoring
of selected antibiotics: distribution and sources of sulfonamides, trimethoprim,
and macrolides in Japanese rivers. Sci Total Environ 2011;409:530512.
Nakada N, Shinohara H, Murata A, Kiri K, Managaki S, Sato N, et al. Removal of selected
pharmaceuticals and personal care products (PPCPs) and endocrine-disrupting
chemicals (EDCs) during sand ltration and ozonation at a municipal sewage treatment
plant. Water Res 2007;41:437382.
Segura PA, Franois M, Gagnon C, Sauv S. May; EHP. Review of the occurrence of
anti-infectives in contaminated wastewaters and natural and drinking waters.
Environ Health Perspect 2009;117:67584.
Sim W-J, Lee J-W, Lee E-S, Shin S-K, Hwang S-R, Oh J-E. Occurrence and distribution of
pharmaceuticals in wastewater from households, livestock farms, hospitals and
pharmaceutical manufactures. Chemosphere 2011;82:17986.
Spongberg AL, Witter JD. Pharmaceutical compounds in the wastewater process stream
in Northwest Ohio. Sci Total Environ 2008;397:14857.
Suzuki S, Hoa PTP. Distribution of quinolones, sulfonamides, tetracyclines in aquatic environment and antibiotic resistance in Indochina. Front Microbiol 2012;3. http://dx.doi.org/
10.3389/fmicb.2012.00067. [article 67, 18].
Takasu H, Suzuki S, Reungsang A, Viet PH. Fluoroquinolone (FQ) contamination does
not correlate with occurrence of FQ-resistant bacteria in aquatic environments of
Vietnam and Thailand. Microbes Environ 2011;26:13543.
Watkinson AJ, Murby EJ, Costanzo SD. Removal of antibiotics in conventional and advanced
wastewater treatment: implications for environmental discharge and wastewater
recycling. Water Res 2007;41:416476.
WHO. http://www.who.int/healthinfo/global_burden_disease/estimates_regional/en/
index.html2012.
Xu W, Zhang G, Li X, Zou S, Li P, Hu Z, et al. Occurrence and elimination of antibiotics at
four sewage treatment plants in the Pearl River Delta (PRD), South China. Water
Res 2007;41:452634.
Ye Z, Weinberg HS, Meyer MT. Trace analysis of trimethoprim and sulfonamide,
macrolide, quinolone, and tetracycline antibiotics in chlorinated drinking water
using liquid chromatography electrospray tandem mass spectrometry. Anal Chem
2006;79:113544.