Article

pubs.acs.org/est

Antibiotics in Drinking Water in Shanghai and Their Contribution to

Antibiotic Exposure of School Children
Hexing Wang,, Na Wang,, Bin Wang, Qi Zhao, Hong Fang, Chaowei Fu, Chuanxi Tang,
Feng Jiang, Ying Zhou,*, Yue Chen, and Qingwu Jiang

Key Laboratory of Public Health Safety of Ministry of Education, School of Public Health, Fudan University, Shanghai 200032,
China

Minhang District Center for Disease Control and Prevention, Minhang District, Shanghai 201101, China

Changning District Center for Disease Control and Prevention, Changning District, Shanghai 200051, China

School of Epidemiology, Public Health, and Preventive Medicine, Faculty of Medicine, University of Ottawa, Ottawa, Ontario
K1H8M5, Canada
S Supporting Information
*

ABSTRACT: A variety of antibiotics have been found in

aquatic environments, but antibiotics in drinking water and
their contribution to antibiotic exposure in human are not
well-explored. For this, representative drinking water samples
and 530 urine samples from schoolchildren were selected in
Shanghai, and 21 common antibiotics (ve macrolides, two lactams, three tetracyclines, four uoquinolones, four
sulfonamides, and three phenicols) were measured in water
samples and urines by isotope dilution two-dimensional
ultraperformance liquid chromatography coupled with highresolution quadrupole time-of-ight mass spectrometry.
Drinking water included 46 terminal tap water samples from dierent spots in the distribution system of the city, 45 bottled
water samples from 14 common brands, and eight barreled water samples of dierent brands. Of 21 antibiotics, only orfenicol
and thiamphenicol were found in tap water, with the median concentrations of 0.0089 ng/mL and 0.0064 ng/mL, respectively;
only orfenicol was found in three bottled water samples from a same brand, with the concentrations ranging from 0.00060 to
0.0010 ng/mL; no antibiotics were found in barreled water. In contrast, besides orfenicol and thiamphenicol, an additional 17
antibiotics were detected in urine samples, and the total daily exposure doses and detection frequencies of orfenicol and
thiamphenicol based on urine samples were signicantly and substantially higher than their predicted daily exposure doses and
detection frequencies from drinking water by Monte Carlo Simulation. These data indicated that drinking water was
contaminated by some antibiotics in Shanghai, but played a limited role in antibiotic exposure of children.

INTRODUCTION
Antibiotics have been extensively used to prevent or treat
bacterial infections in human and veterinary medicine and to
promote growth in animal husbandry and aquaculture for
nearly one hundred years.1 Because a considerable proportion
of antibiotics used in human and animal would be excreted in
urine and faeces as unchanged and active species,2 and
wastewater treatment plants (WWTPs), hospitals, and livestock
farms do not always have enough capacity of removing
antibiotics in their euent, sludge, or manure, antibiotics
have contaminated aquatic environments.3 Due to their
potential threats to aquatic ecological environment and
human health, antibiotics have been thought as one group of
emerging environment contaminants.4 Of more concern is that
the adverse consequence of antibiotics in aquatic environment
is likely to be far-reaching beyond bacterial resistance.5
Antibiotic exposure from aquatic environment might result in
a systematic eect on human body physiology by gut
2016 American Chemical Society

microbiota, which has been linked to many diseases related

to immune and metabolism.6 Given that there are more than
100 antibiotics used in humans and animals79 and nearly 70
antibiotics have been found in surface water and sediment,3,10
the concern of antibiotics in drinking water as a potential
exposure source is rising. In previous studies, however, only a
few categories of antibiotics were investigated in drinking
water,11,12 and their contribution to the overall exposure of
human to antibiotics from various sources is not well-explored.
Shanghai is the largest Chinese city located in the estuary of
the Yangtze River in East China with a relatively high
economical development level. Due to its dense population
and intensive livestock breeding industry, Shanghai was
Received:
Revised:
Accepted:
Published:
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November 22, 2015

January 26, 2016
February 5, 2016
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were kept in an air conditioned room set at 20 C and were

analyzed within 1 week.
Analysis of Antibiotics in Drinking Water. A total of 21
common antibiotics were selected from six categories, including
ve macrolides (azithromycin, clarithromycin, erythromycin,
roxithromycin, and tylosin), two -lactams (cefaclor and
ampicillin), three tetracyclines (chlortetracycline, tetracycline,
and oxytetracycline), four uoquinolones (ciprooxacin,
ooxacin, enrooxacin, and noroxacin), four sulfonamides
(sulfamethazine, sulfamethoxazole, sulfadiazine, and trimethoprim), and three phenicols (chloramphenicol, orfenicol, and
thiamphenicol). Among them, azithromycin, clarithromycin,
roxithromycin, cefaclor, and chloramphenicol are only used as
human antibiotics; tylosin, chlortetracycline, enrooxacin, and
orfenicol are only used as veterinary antibiotics; erythromycin
and ampicillin are preferred as human antibiotics; the remaining
ten antibiotics are preferred as veterinary antibiotics.
A method based on the isotope dilution two-dimensional
UPLCQ/TOF MS was used to measure 21 antibiotics in
drinking water. The sample pretreatment followed an analytical
method described previously.17 Briey, 2 mL of 2.5 g/L
Na2EDTA and 13 stable isotope labeled internal standards were
added to each 500 mL of tap water sample and then was
adjusted to pH 3 and extracted using 200 mg HLB solid-phase
extraction cartridges. During analysis by two-dimensional
UPLCQ/TOF MS, 21 antibiotics were divided into two
groups: one group was consisted of three phenicols and another
group was consisted of remaining 18 antibiotics. Antibiotics in
the former group were analyzed in negative-ion mode, and
those in the latter group were analyzed in positive-ion mode.
The interbatch recoveries of three phenicols varied between
81.7% and 103.7%, with the interbatch relative standard
deviations ranging from 4.2% to 5.9%. The interbatch
recoveries of 18 antibiotics varied between 83.6% and
109.6%, with the interbatch relative standard deviations ranging
from 8.2% to 12.8%. Details of analytical method were provided
in Supporting Information.
Predication of Antibiotic Exposure from Drinking
Water. Daily Exposure Dose. Using concentrations of
antibiotics in drinking water, daily drinking water consumption,
and body weight, the daily exposure dose from drinking water
was estimated in children. The calculation formula was as
C Vd
(Ed: estimated daily exposure dose from
follows: Ed = M d 1000

expected to have a high antibiotic contamination level in

aquatic environment.13 A previous study measured 18 antibiotics in urine from more than 1000 children living in East
China and found that children carried a heavy antibiotic body
burden, but the role of antibiotics from drinking water in
human antibiotic exposure remains unclear.14 In this study, we
investigated 21 antibiotics in drinking water and in urine
samples from school children in Shanghai by isotope dilution
two-dimensional ultra-performance liquid chromatography
coupled with high-resolution quadrupole time-of-ight mass
spectrometry (UPLCQ/TOF MS), estimated the antibiotic
exposure based on drinking water and urine, and made an
assessment for antibiotic contamination in drinking water and
its contribution to overall antibiotic exposure in children.

MATERIALS AND METHODS

Collection of Tap, Bottled, and Barreled Water. Given
that the existing 14 subway lines in Shanghai can almost cover
the entire city, a sampling method based on the stations along
subway lines was employed. To collect representative terminal
tap water samples of Shanghai, besides two sampling sites
respectively set at two end stations of each subway line, we
evenly distributed a suitable number of other sampling sites in
the stations between two ends of each subway line. The details
of subway network map were provided in the ocial Web site
of Shanghai Metro.15 A total of 46 sampling sites were set along
all the 14 subway lines, and the collection was performed in
June 2015.
There are two main water sources for tap water in Shanghai:
the Qingcaosha reservoir in the Yangtze estuary and the upper
reach of the Huangpu River.16 The former is on the north side,
and the latter is on the south side of Shanghai City. It was
inferred that the tap water in the north part of Shanghai mainly
originates from the Qingcaosha reservoir, and the tap water in
the south part of the city mainly originates from the upper
reach of Huangpu River; the tap water in the middle part of the
city may be a mixture from the two water sources. Based on the
subway network map, two virtual lines were drawn to evenly
divide the city into three parts of north, middle, and south, in
which tap water had dierent water sources, and 15, 16, and 15
of 46 selected sampling sites were located in the north, middle,
and south parts of the city, respectively.
A total of 1 L of terminal tap water was collected at each
sampling site. Water samples were collected in glass bottles
previously cleaned by pure water and acetone. To avoid
possible contamination during water sampling, we turned on
the water taps completely to release water for at least 5 min,
and the glass bottles were cleaned at least ve times by tap
water before the formal water collection. After tap water
samples were collected in glass bottles, about 50 mg of ascorbic
acid was immediately added to quench the residual chlorine in
tap water to prevent the antibiotics from degrading.17 Tap
water sampling was performed by ve technicians in two
consecutive days, and each technician carried one water eld
blank to monitor possible interferences during the entire
sampling process.
A total of 45 bottled water samples from 14 dierent brands
were conveniently purchased from super markets in Shanghai,
and at least three bottles were collected for each brand. A total
of eight barreled water samples of dierent brands were
purchased from commercial agents in Shanghai. A single 25 L
barrel of water was collected for each brand. All water samples

drinking water, g/kg/day; Cd: antibiotic concentration in

drinking water, ng/mL; Vd: daily drinking water consumption,
mL/day; Mb : body weight, kg). After the probability
distributions of antibiotic concentrations in drinking water,
body weight, and daily consumption volume of drinking water
were obtained, the probability distribution of daily exposure
doses of antibiotics from drinking water was predicted by
10 000 repetitions of a Monte Carlo simulation using the @
Risk software (version 6.1.1, Palisade Corporation). The
probability distributions of body weight (kg) were dened as
the Weibull distribution with a scale of 1.8485 and a shape of
19.985 for boys and the Weibull distribution with a scale of
1.7948 and a shape of 17.186 for girls by the @Risk software.
The probability distributions of daily consumption volume of
drinking water were set as the normal distribution with a mean
of 773 and a standard deviation of 19 for boys and the normal
distribution with a mean of 622 and a standard deviation of 16
for girls according to a study conducted in 1454 children aged
817 years in Shanghai.18

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Table 1. Detection Frequency and Concentration (ng/mL) of Phenicols in Tap Water and Urines
antibiotics

orfenicol
chloramphenicol
thiamphenicol
a

tap water (n = 46)

urine (n = 530)

n (%)a

average

median (minimummaximum)

n (%)a

46 (100)
0
46 (100)

0.011

0.0089 (0.000820.024)
b
0.0064 (0.000840.023)

181 (34.2)
120 (22.6)
32 (6.0)

0.0076

percentiles

max

50th

70th

75th

95th

99th

0.05

0.06

0.33
0.56
0.02

2.74
32.98
0.06

67.71
157.38
0.88

Positive detection (detection frequency, %). b: less than LOD.

Detection Frequency. Based on concentrations of antibiotics

in tap water, daily drinking water consumption, body weight,
and daily urine output by body weight, urinary concentrations
of antibiotics from drinking water was estimated. The
C V P
calculation formula was as follows: Cu = dM d V (Cu:
b

UPLCQ/TOF MS in negative-ion mode. The interbatch

recoveries of three phenicols varied between 86.1% and 110.2%,
with the interbatch relative standard deviations ranging from
3.8% to 9.3%. Details of analytical method for three phenicols
were provided in the Supporting Information.
Total Daily Exposure Dose. The total daily exposure dose
was calculated using the following formula frequently used in
C M
1
previous daily intake evaluation studies:21,22 Eu = Ca Mc P

estimated urinary concentration in subjects, ng/mL; Cd, Vd,

and Mb were same as mentioned above; Vu: daily urine output
by body weight, ml/kg/day; P: antibiotic excretion proportion
in urine as unchanged and glucuronide-conjugated species
(Table S1)). Because the experimental excretion proportions of
tylosin, enrooxacin, and orfenicol in urine was unavailable in
humans, the urinary excretion proportion from pigs was used
here.13 After the probability distributions of antibiotic
concentrations in drinking water, body weight, daily consumption volume of drinking water, and daily urine output by
body weight were obtained, the probability distribution of
urinary antibiotic concentrations from drinking water was
predicted by 10 000 repetitions of a Monte Carlo simulation to
obtain its probability distribution using the @Risk software.
The probability distribution of daily urine output by body
weight (ml/kg/day) was set as the normal distribution with a
mean of 22.2 and a standard deviation of 2.0.19 Detection
frequency of urinary antibiotics from drinking water in children
was dened as the proportion of predicted urinary concentrations greater than their limits of detection (LODs) among all
predicted urinary concentrations.
Predication of Total Antibiotic Exposure in Children.
Selection of School Children. To better represent children in
Shanghai, we selected two elementary schools in, respectively,
the urban and suburban parts of the city in 2013.14 The urban
school is located in the middle of the city, and the suburban
school is located in the south part of the city. After three or four
classes were randomly selected from the third, fourth, and fth
grades in each school, a total of 530 Han children aged 811
years were included in this study. Of them, 222 came from the
urban school and 308 came from the suburban school; 274
were boys and 256 were girls; 530 children provided rstmorning-urine samples, and 518 children participated in
routine physical examination. The study was reviewed and
approved by the Institutional Review Board of Fudan
University.
Analysis of Antibiotics in Urines. Total urinary concentrations (free and conjugated) of 21 antibiotics were
determined by the isotope dilution two-dimensional UPLC
Q/TOF MS, based on an analytical method modied from that
previously established in our lab.20 Except for three phenicols,
the analysis of 18 antibiotics and creatinine in urine were
described previously.14 For three phenicols, the analytical
method was briey provided as follows: after an aliquot (1.0
mL) of urine was spiked with isotope-labeled chloramphenicol
and hydrolyzed by -glucuronidase, the mixture was puried by
solid-phase extraction and analyzed by two-dimensional

(Eu: estimated daily exposure dose, g/kg/day; Ca: urinary

antibiotic concentration, g/L (equivalent to ng/mL); Cc:
urinary creatinine concentration, mmol/L; Mc: daily output of
urinary creatinine, mmol/day; Mb and P were same as
mentioned above). The daily output of urine creatinine was
predicted by a height-based regression equation based on a
population of 454 healthy children aged 318 years: Mc =
0.0102 Hb 0.6854 with a determination coecient of 0.87
and a p-value less than 0.0001 (Hb: standing height, cm).23
Statistical Analysis. Given that there was lack of specic
individual and population-based data on daily consumption
amount of tap, bottled, and barreled water in Chinese children,
the concentrations and probability distributions of antibiotics
with the highest contamination level among tap, bottled, or
barreled water was assumed as those in drinking water to avoid
possible underestimation of exposure level from drinking water.
To check the validity of prediction by Monte Carlo Simulation,
we compared the average daily dose and urinary concentration
based on average body weight (35.7 kg for girls and 38.0 kg for
boys), average daily drinking water consumption (773 mL/day
for boys and 622 mL/day for girls), average antibiotic
concentration in drinking water, and average daily urine output
by body weight (22.2 mL/kg/day) with those values predicted
by the Monte Carlo simulation. In terms of exposure dose and
urinary detection frequency, antibiotic exposure predicted from
drinking water by Monte Carlo simulation was compared with
those based on urine to explore the role of antibiotics from
drinking water in total antibiotic exposure. Analysis of variance
was used to test for antibiotic concentration dierences in tap
water samples by their geographic locations in north, middle,
and south parts of the city. Rank and chi-square test were used
to test for the dierences of concentration distributions and
detection frequencies of urinary antibiotics between the urban
and suburban schools, respectively. All statistical analyses were
performed using the statistical software packages SPSS (version
17; SPSS, Inc., Chicago, IL). A p-value of <0.05 was considered
statistically signicant.

RESULTS
Of 21 antibiotics, only two phenicols (orfenicol and
thiamphenicol) were detected in tap water samples, with the
median concentrations of 0.0089 and 0.0064 ng/mL,
respectively (Table 1); only orfenicol was found in three
bottled water samples from a same brand with the
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Figure 1. Probability distribution of predicted daily exposure dose (DED) (A1, A2, B1, and B2) and urinary concentrations (C1, C2, D1, and D2) of
orfenicol and thiamphenicol from drinking water by Monte Carlo simulation (n = 10000). In A1, A2, B1, and B2, the red section of the top bar
indicates the 99th percentile of predicted daily exposure doses; in C1, C2, D1, and D2, the red fragmentation of the top bar indicates the proportion
of predicted urinary concentrations greater than the limits of detection (LODs) among all predicted urinary concentrations; both LODs of two
phenicols are 0.01 ng/mL.

found in urine samples, with the dierent detection frequencies

from 0.4% (tetracycline) to 25.7% (sulfamethazine) (Table S2).
Because the contamination level of orfenicol and
thiamphenicol in tap water was higher than that in bottled or
barreled water, their concentrations and probability distributions in tap water were assumed as those in drinking water.
According to the @Risk software, the probability distributions
of orfenicol concentration in tap water were dened as the
Gumbel distribution with a location of 8.0492 and a scale of

concentrations of 0.00060, 0.00076, and 0.0010 ng/mL,

respectively. No antibiotics were detected in barreled water
samples. A total of three phenicols (orfenicol, thiamphenicol,
and chloramphenicol) were found in urine samples with varying
detection frequencies from 6.0% to 34.2%, and chloramphenicol showed a higher concentration than other two phenicols
(Table 1). Besides orfenicol and thiamphenicol, an additional
17 of 19 antibiotics, except for roxithromycin and tylosin, were
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Table 2. Comparison of Antibiotic Exposure between Values Predicted from Drinking Water by Monte Carlo Simulation and
Based on Urine
antibiotics

sex

predicted exposure from drinking water (n = 10000)

n (%)a

orfenicol
thiamphenicol

boy
girl
boy
girl

950 (9.5)
580 (5.8)
140 (1.4)
60 (0.6)

percentilesb

total exposure based on urine (n = 518)

maximum

50th

75th

95th

99th

0.00046
0.00040

0.00064
0.00056
0.00048

0.0013
0.0013
0.0012
0.0008

n (%)c
95 (35.8)
80 (31.6)
15 (5.7)
17 (6.7)

percentilesb

maximum

50th

75th

95th

99th

0.039
0.031

0.12
0.16
0.00044
0.0013

0.26
0.38
0.0022
0.023

0.38
0.40
0.0050
0.036

Predicted positive detection (predicted detection frequency, %). bSelected percentiles of daily exposure dose (g/kg/day). cActual positive
detection (actual detection frequency, %). d : Less than LOD.

4.3112, and those of thiamphenicol were dened as the Gumbel

distribution with a location of 5.6133 and a scale of 3.2585.
Figure 1 shows the probability distributions of daily exposure
doses and urinary concentrations of orfenicol and thiamphenicol predicted from drinking water by Monte Carlo simulation.
The predicted urinary detection frequencies were 9.5% in boys
and 5.8% in girls for orfenicol and 1.4% in boys and 0.6% in
girls for thiamphenicol. The selected percentiles of predicted
daily exposure doses and urinary concentrations were,
respectively, listed in Tables S3 and S4. The averages of
predicted daily exposure doses of orfenicol were 0.00022 g/
kg/day in boys and 0.00019 g/kg/day in girls, and those of
thiamphenicol were 0.00016 g/kg/day in boys and 0.00014
g/kg/day in girls. The averages of predicted urinary
concentrations of orfenicol were 0.0056 ng/mL in boys and
0.0049 ng/mL in girls and those of thiamphenicol were 0.0034
ng/mL in boys and 0.0030 ng/mL in girls. These values were
similar to those based on average values of body weight, daily
drinking water consumption, concentrations of two phenicols
in drinking water, or daily urine output by body weight (Table
S5).
The daily exposure doses and urinary detection frequencies
of orfenicol and thiamphenicol based on urine were compared
with those predicted from drinking water, and the exposure
level of orfenicol based on urine showed a higher level than
that of thiamphenicol (Table 2). The detection frequencies of
two phenicols based on urine were 3 to 11 times higher than
those predicted from drinking water. The selected percentiles
of daily exposure doses based on urine were 260 to 680 times
higher than those from drinking water for orfenicol and were
4 to 45 times for thiamphenicol. Besides two phenicols, the
daily exposure doses of other 19 antibiotics were also estimated
(Table S6). The estimated daily exposure dose varied greatly by
antibiotics, and some extremes exceeded 1 mg/kg/day for some
human antibiotics. Figure 2 shows that at least one antibiotic
was found in 78.3% of children. Of them, 56.4% of children had
a sum of estimated daily exposure doses of 21 antibiotics that
were less than 1.0 g/kg/day, 10.3% between 1.0 g/kg/day
and 10.0 g/kg/day, and 11.6% greater than 10 g/kg/day.
Moreover, the concentrations of orfenicol and thiamphenicol in tap water were similar among in the south, middle, and
north parts of Shanghai (Table 3). Urinary concentrations and
detection frequencies of thiamphenicol in children living in the
suburb located in the south part of Shanghai were found to be
lower than those of children living in the downtown in the
middle part of the city, but this phenomenon did not exist for
orfenicol (Figure 3).

Figure 2. Frequency distribution of the sum of daily exposure dose

estimates of all antibiotics (n = 518).

Table 3. Concentrations of Two Phenicols in Tap Waters by

Their Geographic Locations in North, Middle, and South
Parts of Shanghai

location

orfenicol

north (n = 15)
middle (n = 16)
south (n = 15)
p-valueb

0.011 0.0059
0.011 0.0055
0.0096 0.0049
0.726

thiamphenicol
a

0.0081 0.0044
0.0084 0.0053
0.0061 0.0036
0.324

Mean SD (ng/mL) . bAnalysis of variance.

DISCUSSION
In this study, by screening 21 common antibiotics in
representative drinking water samples using two-dimensional
UPLC-Q/TOF MS, two phenicols (orfenicol and thiamphenicol) were detected in tap water, orfenicol was detected
in some bottled water, and no antibiotics were detected in
barreled water. To our knowledge, this study is the rst time to
comprehensively screen antibiotics in bottled and barreled
water. In contrast, besides orfenicol and thiamphenicol, an
additional 17 antibiotics were detected in urine samples with
varying detection frequencies, and the predicted daily exposure
of orfenicol and thiamphenicol from drinking water by the
Monte Carlo simulation were signicantly lower than those
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comparable to those reported for the surface water of Shanghai

(0.91110 ng/L for orfenicol and 0.4589.5 ng/L for
thiamphenicol).29 Florfenicol was also found in some bottled
water produced from other water sources outside Shanghai in
China. Besides conventional water treatment techniques, some
advanced techniques, such as ozone disinfection and activated
carbon ltration, have been introduced in water plants in
Shanghai. These results suggest that water-treatment processing
in Shanghai can eciently remove most antibiotic residues in
source water but not phenicols in the basis of current advanced
techniques used.
Drinking Water and Other Possible Sources of
Antibiotic Exposures in Children. Of 21 selected antibiotics,
only orfenicol and thiamphenicol were detected in drinking
water. Their level of antibiotic contamination was higher in tap
water than that in bottled or barreled water. In the estimation
of daily exposure dose from drinking water, using tap water
measurements could result in an overestimation of true dailyexposure dose of orfenicol and thiamphenicol from drinking
water. However, their predicted daily exposure dose and
detection frequency were still signicantly and substantially
lower than those based on the urine levels. Furthermore, the
concentrations of orfenicol and thiamphenicol in tap water
were also found not to change with those in urines by sampling
sites. These data collectively indicated that drinking water was
not an important source of antibiotic exposure in children.
Generally, there are three sources for exposure of children to
antibiotics: direct utilization, drinking water, and food. After
drinking water was ruled out as a primary antibiotic exposure
source, the primary exposure sources were inferred to be direct
utilization and contaminated food. Antibiotics can be divided
into two groups based on their application objects: one group is
only used or preferred in animals, and another group is only
used or preferred in humans. The exposure sources of children
to antibiotics might depend on their applications; that is,
mainly via contaminated food for antibiotics only used or
preferred in animals and mainly via direct utilization for
antibiotics only used or preferred in humans. Moreover, given
that antibiotics only used or preferred in animals are used to
prevent or treat diseases or as growth promoters in breeding
industry,30 their primary exposure source was likely to be meat
foods, such as those derived from livestock, poultry, and
aquaculture.
Comparative Analysis. Only a few studies reported the
contamination of antibiotics in tap water, which partially
supported our results.12,31 Leung et al. found 11 common
antibiotics in tap waters from 13 cities in China, such as
thiamphenicol, sulfamethoxazole, sulfamethazine, azithromycin,
trimethoprim, tylosin, and dimetridazole.31 Of them, thiamphenicol were detected in the tap water of Hangzhou in
Zhejiang Province and Nanjing in Jiangsu Province, and
sulfamethoxazole, sulfamethazine, and dimetridazole were
detected in Shanghai. Some antibiotics were also found in tap
water in the United States and some countries in Europe and
Asia, such as sulfamethoxazole, sulfadiazine, trimethoprim, and
azithromycin.11,32,33 The detection of antibiotics might be
related to antibiotic contamination extent in source water,
water-treatment techniques, and sampling seasons.30,34
However, there have been no reports about orfenicol
contamination in tap water. Several studies reported its
contamination of surface water in other areas than Shanghai
in China,3537 but few in other countries. For example,
orfenicol were detected in Jiulong River in Fujian Province,37

Figure 3. Comparison of urinary concentration (a) and detection

frequency (b) of orfenicol and thiamphenicol in children from
between the downtown (n = 222) and suburban (n = 308) parts of
Shanghai (The dierence of urinary concentration and detection
frequency were analyzed by rank sum test and Chi square test,
respectively; * indicates p-value of <0.001).

based on urine. The average values of daily exposure dose and

urinary concentration for two phenicols predicted by the
Monte Carlo Simulation were well-matched with those based
on the average value of each variable. These ndings indicated
that drinking water was contaminated by some antibiotics in
Shanghai, but played a limited role in the total antibiotic
exposure in children.
Sources of Antibiotics in Drinking Water. Generally, the
primary antibiotic source in aquatic environment is from the
excretion of antibiotics used in animals and human as
unchanged or conjugated species in urine or feces.13 Direct
discarding during the use or production of antibiotics is another
possible pathway.24 Antibiotics in aquatic environment have to
survive a series of drinking water treatment processes before
residue in drinking water. Water treatment processing includes
conventional techniques, such as coagulation, sedimentation,
ltration, and chlorine disinfection, and relatively advanced
techniques, such as reverse osmosis, ozone disinfection, and
activated carbon sorption. The removal eciencies of these
treatment techniques have been tested for some antibiotics.2527 In general, the conventional water-treatment
techniques, except for chlorine disinfection, did not eciently
remove antibiotics, but the advanced techniques did, such as for
amoxicillin, tetracycline, oxytetracycline, sulfamethoxazole,
sulfamethazine, trimethoprim, and erythromycin.26,27 However,
there were no reports about the removing eciency of
phenicols by these technologies.
A variety of antibiotics have been detected in the surface
water of Yangtze estuary and Huangpu River, which are the
primary water sources of tap water in Shanghai.28,29 Among
them, phenicols, sulfonamides, trimethoprim, and macrolides
were frequently detected, followed by tetracyclines and
uoquinolones.29 However, only two phenicols (orfenicol
and thiamphenicol) were found in terminal tap water in
Shanghai in our study, and their concentration ranges were
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the reservoirs in North China,36 and some river and pond
waters in Jiangsu Province.35 Given that phenicols might not be
eciently removed by current water-treatment techniques, and
water-treatment processes were similar among water plants in
China, it might be common for orfenicol contamination in tap
water in China.
Total Antibiotic Exposure and Its Implications for
Future Studies. The sum of daily exposure dose of all 21
antibiotics showed that more than half of children were exposed
to antibiotics around the 1.0 g/kg/day level. Compared to the
dose level around or above the mg/kg/day level resulting from
their application, this can be dened as a low-dose level. The
risk factors related to antibiotic exposure are relatively stable in
a certain period, such as antibiotic use in human and veterinary
medicine, occurrence of infectious diseases, dietary habits, and
antibiotic contamination extent of aquatic environment and
food; therefore, a long-term exposure to low-dose antibiotics
was inferred to be the primary exposure mode in children. The
occurrence of low-dose antibiotics in childrens bodies could
originate from the exposure to low-level residues of antibiotics
in food or the metabolic gradients of exposure to high-level
antibiotics, such as from antibiotic use.
It is noteworthy that current studies about potential health
hazards of antibiotics are based on antibiotic use or relevant
high-dose exposure at the mg/kg/day level, and most focused
on the short-term eects of high-dose antibiotic exposures,38
except that bacterial resistance has only been investigated under
a sublethal concentration of antibiotics.39 Besides bacterial
resistance, however, the possible health consequences of
antibiotic exposure also include direct side eects, such as the
eects on growth and hematopoietic function of children,40,41
and health outcomes related to the human microbiome, such as
psoriasis, inammatory bowel disease (IBD), asthma, diabetes,
obesity, cardiovascular disease, and colorectal cancer.5,6 Because
a signicant dierence exists between two kinds of exposure
modes (i.e. short-term exposure to high-dose antibiotics and
long-term exposure to low-dose antibiotics), more studies are
needed to clarify the eects of the latter exposure mode on
human health.
Strengths and Limitations of the Study. This study
comprehensively assessed the antibiotic contamination of
drinking water in Shanghai by sampling representative terminal
tap, bottled, and barreled water. In the prediction of antibiotic
exposure in children from drinking water, the Monte Carlo
simulation considering the probability distributions of variables
provided more accurate estimates, compared to conventional
estimation method based only on mean or median. Moreover,
total antibiotic exposure in children was accurately estimated by
the biomonitoring data of urinary antibiotics, and daily urinary
creatine output, a key variable of estimating total antibiotic
exposure, was predicted for each child by their body heights,
which could reduce the estimation uncertainty compared to the
sex- and age-specic cut-o values as used in other studies.21,22
There were several limitations in the interpretation of the
results. First, the assumption of antibiotic contamination level
in tap water as that in drinking water and the unconsidered
eects of some cooking processes on the degradation of
antibiotics, such as boiling, would overestimate the true daily
antibiotic exposure dose from drinking water, but this further
enhanced the inference that drinking water played a limited role
in total antibiotic exposure in children. Second, in this
estimation of total antibiotic exposure based on urine, the
ratio of antibiotic urine excretion rate to urinary creatinine rate

was assumed to be constant during the day, but the antibiotic

urine excretion rate might uctuate throughout the day due to
discontinuous antibiotic exposure and their rapid metabolism
rate;42 thus, daily variation might exist in the ratio. Third, the
urinary excretion proportion of low-dose antibiotics was also
assumed to be equal to that of high-dose antibiotics in the
estimation of total antibiotic exposure, but the excretion
proportions of some antibiotic in urine were dose-dependent.43,44 Due to a lack of pharmacokinetic data for low-dose
antibiotic exposure, a proportion of antibiotic excretion in urine
based on the pharmacokinetics at high-dose antibiotic exposure
was used identically.

ASSOCIATED CONTENT

S Supporting Information
*

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.est.5b05749.
Additional text and six tables providing details of
analytical method of antibiotics in drinking water and
urine, urinary excretion proportions of 21 antibiotics as
unchanged and glucuronide-conjugated species, predicted daily exposure doses and urinary concentrations
of orfenicol and thiamphenicol from drinking water,
urinary detection frequencies and concentrations of 18
antibiotics in school children, urinary concentrations and
daily exposure doses of two phenicols based on average
values of each variable, and daily exposure dose estimates
of 21 antibiotics based on urine. (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel and fax: 086 021 54237565; e-mail: yingchou@fudan.edu.

cn.
Author Contributions

These authors contributed equally to this work.

Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS
This work was supported by Research Initiation Funds for New
Teacher of Fudan University (no. JJF201204), the Natural
Science Foundation of China (no. 81373089), 985 Innovation
Platform Project for Superiority Subject of Ministry of
Education of China (no. EZF201001), and the Scientic
Research Foundation for Health Field, National Health and
Family Planning Commission of China (no. 201202012).

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