Environmental Toxicology and Chemistry, Vol. 25, No. 1, pp.

265–271, 2006
q 2006 SETAC
Printed in the USA
0730-7268/06 $12.00 1 .00

ECOTOXICOLOGICAL RISK OF PHARMACEUTICALS FROM WASTEWATER

TREATMENT PLANTS IN KOREA: OCCURRENCE AND TOXICITY TO
DAPHNIA MAGNA

GUK H. HAN, HOR G. HUR, and SANG D. KIM*

Department of Environmental Science and Engineering, International Environmental Research Center, Gwangju Institute of Science and
Technology, 1 Oryong-dong, Buk-gu, Gwangju 500-712, Korea

( Received 17 March 2005; Accepted 16 June 2005)

Abstract—The overall ecotoxicological effect of pharmaceutically active compounds (PhACs) detected in the effluents of Korean
wastewater treatment plants (WWTPs) to Daphnia magna was investigated using biological and chemical analyses. The bioassay
results showed median lethal concentrations and no-observed-effect concentrations ranging from a few to tens of ppm levels for
nine PhACs in 48-h acute and 21-d chronic tests. The mixture effects of pharmaceuticals also were examined by other acute and
chronic tests, which showed no significant toxicity despite a slightly increased combined effect of approximately twofold. The
residual concentrations of nine PhACs were analyzed at concentrations ranging from 10 ng/L to 89 mg/L in the influents and from
10 ng/L to 11 mg/L in the effluents from four metropolitan cities in South Korea between January and November of 2004. Through
repeated investigations of the influents and the effluents from different WWTPs, relatively higher removal efficiencies (23.9–91.3%)
compared with those of previous surveys performed in other countries were observed for most pharmaceuticals, with the exception
of acetaminophen (8.7%). The present study showed no significant risk effects of the effluents from WWTPs containing pharma-
ceuticals (i.e., hazard quotient , 1), even at the 95th percentile contamination range, although a risk assessment factor of 1,000
was applied. Therefore, it can be concluded that the potential risk of pharmaceuticals should be monitored carefully with more
bioassay data, because many uncertainties still exist in the determination and toxicity of metabolites in water environments. No
significant risk was observed, however, from the selected PhACs in the effluents from WWTPs discharged into surface waters.

Keywords—Pharmaceuticals Wastewater treatment plants Toxicity Risk assessment

INTRODUCTION aquatic environments [7–13]. Many metabolites, such as hy-

Although large quantities of pharmaceuticals are produced
and used as animal and human medicines, little attention has
been paid to the potential risks of pharmaceuticals as toxic
contaminants in aquatic environments. Pharmaceuticals have
similar physicochemical behaviors and cause biological effects
toward nontarget organisms in the environment (i.e., human
and animals) even at low concentrations [1,2]. Recent studies
have demonstrated that some metabolites are more lipophilic
and more persistent than the original drugs from which they
were derived [3]. These substances are excreted or discarded
from both humans and domestic animals as a mixture of me-
tabolites and unchanged forms via the municipal sewage sys-
tem to rivers, bank filtrates, and groundwaters. This implies
that their parent compounds and metabolites may exist in
drinking water through several drug-fate pathways. The bal-
ances of the influents and effluents of drug residues detected
in wastewater treatment plants (WWTPs) reveal that many
pharmaceuticals are not eliminated completely by traditional
treatment processes (i.e., coagulation, flocculation, and sedi-
mentation) [4].
Recently, major efforts to clarify the ecological influence
and occurrence of pharmaceuticals in the aquatic environment
have been attempted in North American and European coun-
tries [1,3,5,6]. Diclofenac, ibuprofen, and clofibric acid are
medical agents used widely in both human and domestic an-
imal practices. These pharmaceuticals have been detected fre-
quently at concentrations up to several ng/L or mg/L levels in
* To whom correspondence may be addressed (sdkim@gist.ac.kr).
265
droxy- and carboxy-ibuprofen and carboxy-hydrotrophic acid
for ibuprofen [11,12] and 2-(4-chlorophenoxy)-2-methyl pro-
pionic acid for clofibric acid [3], have been found in raw
sewage water and rivers. Ibuprofen and its metabolites are
degraded efficiently by biological treatment in WWTPs,
whereas diclofenac and clofibric acid are degraded only min-
imally by biological reaction [3,5,7]. More than 90% of the
diclofenac in Lake Greifensee, Switzerland, was removed by
photolytic degradation [7]. Some of the other substances de-
tected in rivers include antipyretics (i.e., acetaminophen [5,6]),
b-sympathomimetics [14], antibiotics (e.g., erythromycin
[15,16]), as well as antiepileptics (e.g., carbamazepine
[12,17,18]) and hypolipidemics (i.e., gemfibrozil [5,6,19]). De-
spite many studies, it remains unclear whether the exposure
of aquatic biota to these pharmaceuticals have direct or indirect
effects on the environments. Furthermore, no data are available
from Korea, which uses large quantities of drugs because of
the high population densities and expanding sizes of its cities.
The objectives of the present study were to monitor the
extent of drug residues in Korean WWTPs and to investigate
the ecological risks of target compounds. First, the acute and
chronic toxicity of pharmaceuticals, both singularly and as
mixtures, were evaluated for Daphnia magna. Using ecotox-
icological results, environmental risk assessments were per-
formed for drug residues in Korean rivers receiving waters
from WWTP effluents to predict their potential adverse effects.
MATERIALS AND METHODS
Sample collection
Samples were collected between January and November of
2004 using precleaned, amber-glass containers during periods
266 Environ. Toxicol. Chem. 25, 2006 G.H. Han et al.

Table 1. Overview of pharmaceutically active compounds

Compounds CASa no. Molecular weight Molecular structure Therapeutic use

Diclofenac 15307-79-6 318.1 Nonsteroidal anti-inflammatory

Ibuprofen 15687-27-1 206.3 Nonsteroidal anti-inflammatory

Clofibric acid 882-09-7 214.7 Lipid regulator (active metabolite)

Carbamazepine 298-46-4 236.3 Antiepileptic

Salicylic acid 69-72-7 138.1 Analgesic, antipyretic

Gemfibrozil 25812-30-0 250.3 Lipid regulator

Acetaminophen 103-90-2 151.2 Analgesic antipyretic

Bezafibrate 41859-67-0 361.8 Antihyperlipidemic

Tolfenamic acid 13710-19-5 261.7 Nonsteroidal anti-inflammatory

a CAS 5 Chemical Abstracts Service.

of normal WWTP operation. Big cities, including Seoul, Bus-
an, Gwangju, and Daegu, were selected as study areas because
of their high population densities, which were assumed to have
exposure to large amounts of pharmaceuticals to their aquatic
environments because of the discharge of effluents. Treatments
at all WWTPs consisted of three main steps: Preliminary clar-
ification, final clarification, and an aeration tank. Gwangju2
had additional treatment steps for phosphate removal and de-
nitrification treatment. The WWTP in Busan also was equipped
with sand filtration, followed by an ultraviolet disinfection
step, after the final clarification. Water samples were stored in
an icebox for their return to the laboratory, adjusted to pH less
than 2 by the addition of concentrated HCl, and preserved at
a temperature less than 48C before analysis. Samples were
filtered through glass-fiber filters (0.45 mm) and sucked
through solid-phase extraction cartridges containing 500 mg
of Sep-Pakt Vac 6cc C18 (Waters, Milford, MA, USA) at a
rate of approximately 8 ml/min. Cartridges were dried for 2
h under a moderate stream of nitrogen and then extracted by
elution with three successive 1-ml aliquots of high-perfor-
mance liquid chromatography (HPLC)–grade methanol [20].
After elution, the samples were evaporated to a final 1-ml
volume.
Analytical procedure
All solutions of pharmaceutically active compounds
(PhACs) were prepared in HPLC-grade methanol, purchased
from Sigma-Aldrich Chemical (St. Louis, MO, USA), for anal-
ysis. Chemicals were analyzed by gas chromatography–mass
spectrometry (GC-MS; Shimadzu, Kyoto, Japan) in full-scan
or selected ion-monitoring mode. Separations were carried out
using a DB5-type capillary column (length, 30 m; inner di-
ameter, 0.25 mm; film thickness, 0.25 mm) with helium
(99.999%) as the carrier gas (flow rate, 1.1 ml/min) and the
flow velocity set to 38.1 cm/s in the constant-flow mode. In-
jections were performed in the split mode (purge time, 2 min)
with an injection volume of 1 ml. The GC oven was pro-
grammed as recommended by Ternes et al. [21]. The methyl
esters of the analytes were quantified by monitoring the ions
at m/z (mass to charge ratio) 214 and 242 for diclofenac;
m/z 161 for ibuprofen; m/z 128 for clofibric acid; m/z 80, 109,
and 151 for acetaminophen; m/z 95, 165, and 193 for carba-
mazepine; m/z 92, 120, and 138 for salicylic acid; and m/z
122 and 107 for gemfibrozil. All samples were analyzed by
five-point calibration over the whole procedure. Using this
method, limits of detection of 10 and 250 ng/L were obtained
for the sewage influents and effluents, respectively. The target
pharmaceuticals used in the bioassay experiments were se-
lected with respect to their occurrence, potential ecological
effect, and broad application (Table 1). Individual stock so-
lutions of each target pharmaceutical were prepared by dis-
solving in ethanol for 1 d before use for each experiment, with
the concentration of the solvent in the medium, including the
control groups, being less than 1%.
Test organisms and culture
The test organism used in the present study, D. magna,
was obtained from the Korea Institute of Toxicology (Daejeon,
Occurrence and toxicity of pharmaceuticals from Korean WWTPs
South Korea). Daphnia magna was cultured with Selenastrum
capricornutum and a mixture of yeast, CEROPHYLL t
(Ward’s, Rochester, NY, USA), and trout chow (YCT) supplied
as food. The organisms were cultured and handled according
to the procedures outlined in the U.S. Environmental Protec-
tion Agency (U.S. EPA) manual [22]. Neonates of D. magna
less than 24 h old were used for acute and chronic tests, which
were illuminated with a 16:8-h light:dark photoperiod. Or-
ganisms were not fed during the test periods. Reconstituted
hard water (MgSO4, 9.98 3 1024 M; CaSO4, 6.98 3 1024 M;
NaHCO3, 2.28 3 1023 M; and KCl, 1.07 3 1024 M), with a
hardness of 170 6 5 mg/L (mean 6 SD), an alkalinity of 110
6 5 mg/L as CaCO3 mg/L, and pH 7.8 6 0.2 at 258C through-
out the study.
Single-compound bioassay tests
For 48-h static nonrenewal acute toxicity tests, D. magna
was exposed to a control and eight different concentrations of
PhACs. Four replicates, containing five organisms per 30-ml
test chamber, were assigned to the treatments and control. Test
organisms were fed YCT and S. capricornutum for 2 h before
the test [22]. Mortality was recorded 48 h later.
The 21-d chronic effect of each pharmaceutical was as-
sessed on the reproduction of D. magna in a static renewal
test according to the U.S. EPA guidelines [23]. The exposure
concentrations to D. magna in 21-d chronic tests ranged from
0.1 to 80 mg/L based on the results of the acute toxicity tests
for nine different concentrations and one control. Five repli-
cates, containing one organism per test chamber, were assigned
to each concentration. Organisms were fed every 2 d with
YCT and S. capricornutum, with all test solutions renewed
with fresh culture medium every 2 d. The number of neonates
reproduced and the survival of D. magna were counted and
recorded on each of the 21 d.
Mixture bioassay tests
To elucidate the combined effects of the pharmaceutical
mixtures to which the aquatic ecosystems were exposed, the
toxic unit (TU) concept was used as follows:
concentration (mg/L)
TU 5 (acute test)
LC50
concentration (mg/L)
TU 5 (chronic test)
NOEC
where LC50 is the single-substance concentration causing 50%
mortality when applied alone and NOEC is the no-observed-
effect concentration. The total combined effect can be cal-
culated by summation of each TU.
For mixture acute toxicity tests, the methodology of equi-
toxic addition of chemicals tested was employed. Daphnia
magna was exposed to the control and five TUs, ranging from
0.18 to 1.05, in 48-h static nonrenewal tests. For example, to
prepare 0.6 TU of a three-chemical mixture, 0.2 TU for each
chemical (diclofenac, ibuprofen, and clofibric acid) was com-
bined. Four replicates, containing five organisms per test cham-
ber, were assigned to the treatments and the control. Other
experimental procedures, such as illumination, feeding regime,
and endpoint measurement, were the same as those for a single
compound.
For the mixture chronic toxicity test, D. magna was ex-
posed to less than 1 TU for the five treatments and one control
for 21 d. Ten replicates, containing one organism per test cham-
Environ. Toxicol. Chem. 25, 2006 267
ber, were assigned for each treatment under the same test con-
ditions as used for a single compound.
Statistical analysis
The endpoints of the toxicity tests using D. magna are based
on adverse effects on survival and reproduction. The LC50s
for the acute tests were calculated using regression analysis
following probit analysis. The Dunnett method of pair-wise
separation was used to determine the NOEC in the 21-d chronic
test. The experimental response used in the statistical analysis
was the total number of neonates produced until the end of
the experiment or at the time of death. An animal that died
before reproducing neonates was included in the analysis, with
zero entered as the number of young produced [23].
Risk assessment
Ecological risk assessments were performed to characterize
the degree of contamination and to evaluate the adverse effects
of these chemicals in real aquatic environments [24]. In the
present study, results from the D. magna toxicity tests were
compared to those from the GC-MS analyses of selected com-
pounds in the influents and effluents of the WWTPs. The haz-
ard quotient (HQ) was calculated to evaluate the degree of
risks for each target PhAC, which was mathematically for-
mulated as follows:
EC (Exposure concentration)
HQ 5
TRV
where TRV represents the toxicity reference value (i.e., LC50
or NOEC). A HQ value of less than 1 when the assessment
factor was applied indicates an insignificant risk. In the present
study, we employed a quite moderate assessment factor of
1,000, as recommended by the European Union [25] and the
Organization for Economic Co-operation and Development
[26].
RESULTS AND DISCUSSION
Bioassay tests
Single-compound toxicity. The mortality (LC50) and repro-
duction inhibition (NOEC) were measured in D. magna ex-
posed to nine pharmaceuticals. The results indicated that most
pharmaceuticals tested were toxic and harmful to aquatic or-
ganisms (Table 2). Generally, toxicity levels of chemicals are
classified as follows: very toxic to aquatic organisms for less
than 1 mg/L, toxic to aquatic organisms for 1 to 10 mg/L, and
harmful to aquatic organisms for 10 to 100 mg/L [25]. Com-
paring the measured and predicted LC50s using the Ecological
Structure–Activity Relationships (ECOSAR) model from the
website of the U.S. EPA [1], the toxicity values showed rel-
atively significant differences for high-lipophilic compounds,
including diclofenac, ibuprofen, and tolfenamic acid, than for
the hydrophilic compounds. For diclofenac, Sanderson et al.
[1] used an extremely low octanol–water partition coefficient
(e.g., log Kow of 0.7; in the present study, log Kow of 4.51 was
used), which caused a large difference in the toxicity value.
To investigate the relationship of toxicity with respect to hy-
drophobicity, the LC50 versus log Kow values were plotted, as
shown in Figure 1. This figure indicated a linear relationship
between the toxicity values and hydrophobicities (r2 5 0.79).
That is, the LC50s decreased (i.e., toxicity increases) with
increasing log Kow of the pharmaceuticals. However, the result
for acetaminophen was exceptional, showing high toxicity for
an extremely low log Kow of 0.27.
268 Environ. Toxicol. Chem. 25, 2006 G.H. Han et al.

Table 2. Toxicity values of pharmaceuticals to Daphnia magna in 48-h acute and 21-d chronic tests

Literature review
LC50b NOECd
Chemicals Log Kowa (mg/L) Hazard classc (mg/L) LC50e NOEC

Diclofenac 4.51 80.1 Harmful 10 5057.0 —

Ibuprofen 3.97 132.6 Nontoxic 20 38.0 3f
Clofibric acid 2.57 141.2 Nontoxic 40 293.0 —
Carbamazepine 2.45 111 Nontoxic — 111.0 —
Salicylic acid 2.26 111.7 Nontoxic — 59.0 —
Gemfibrozil 4.77 10.4 Harmful — 6.0 —
Acetaminophen 0.27 20.1 Harmful — 42.0 —
Bezafibrate 4.25 30.3 Harmful — 25.0 —
Tolfenamic acid 5.17 7.4 Toxic — 1.7 —
a Values from Howard and Meylan [33] and Sanderson et al. [1].
b LC50 5 median effective concentration.
c Classified in European Union Directive 93/67/EEC (European Economic Communities) [25] in the
basis of LC50 values.
d NOEC 5 no-observable-effect concentration.
e Values from Sanderson et al. [1] calculated by the structure–activity relationship model using Ecological

Structure–activity relationships (ECOSAR).

f Value from Halling-Sorensen et al. [28].

In the 21-d chronic toxicity tests, the concentration–re-
sponse curves for the single substances were sigmoid but had
different sharpness and shapes. When D. magna was exposed
to 80 mg/L of each PhAC, the mortality rates were 100%. All
compounds showed good reproduction rates at 10 mg/L. The
observed mortality rate with diclofenac was 100% at the rel-
atively low concentration of 40 mg/L. The single chronic tox-
icity results indicated that diclofenac was the most toxic phar-
maceutical in the present study. The mortality by the end of
the experiment never exceeded 10% for the controls. The
NOECs were 10, 20, and 40 mg/L for diclofenac, ibuprofen,
and clofibric acid, respectively. Compared with the acute tox-
icities, the NOECs were not low, as shown by the acute to
chronic ratio (LC50:NOEC), which ranged from 3.5 to 8. This
implies that the tested chemicals (i.e., diclofenac, ibuprofen,
and clofibric acid) show no serious long-term effects toward
aquatic ecosystems [27,28].
The LC50s, even the chronic values (NOECs), were quite
high for single compounds, in the range 10 to 100 mg/L (not
toxic but harmful to aquatic organisms). These values were
5,000- to 100,000-fold higher than the median concentrations
detected in aquatic environments [5]. Accordingly, considering
the presence of low concentrations of these pharmaceutical
residues in real environments, such as rivers and wastewater
effluents containing various PhACs within the range 0.1 to 10
mg/L, we can conclude that the acute effects, or even chronic
effects (NOECs), of single compounds are negligible.
Mixture toxicity. To evaluate the combined effect of mix-
tures, toxicity tests were carried out on D. magna with a com-
bination of the three drugs (diclofenac, ibuprofen, and clofibric
acid). The observed and calculated toxicities, based on the TU
concept, were compared (Fig. 2). At greater than 0.5 TU, the
TU observed in bioassay experiment showed approximately
twofold higher toxicity values than the TU predicted by cal-
culation. For example, addition of the mixture concentration
corresponding to 1.0 TU (i.e., 50% mortality in calculation)
for three pharmaceuticals resulted in 2.0 TU (i.e., an observed
100% mortality) to D. magna. The measured toxicity values

Fig. 2. Comparison of the observed and calculated percentage mor-

Fig. 1. The relationship between the toxicity (median lethal concen-
tration [LC50]) and hydrophobicity (log Kow) for nine pharmaceuticals
in the Daphnia magna 48-h acute toxicity tests. The solid line rep-
resents the linear regression of the data (r2 5 0.79). The closed circle
indicates the data for acetaminophen.
tality based on the toxic unit (TU) in 48-h acute mixture toxicity tests
to Daphnia magna. The dashed line shows a 1:1 slope. The observed
TU was determined from the percentage mortality results, with the
calculated TU indicating the sum of the TUs of individual pharma-
ceuticals based on their median lethal concentrations.
Occurrence and toxicity of pharmaceuticals from Korean WWTPs Environ. Toxicol. Chem. 25, 2006 269

Occurrence of pharmaceuticals from WWTP effluents

Fig. 3. Comparison of the observed and calculated percentage inhi-
bitions, based on the toxic unit (TU), in 21-d chronic mixture toxicity
tests to Daphnia magna. The dashed line shows a 1:1 slope. The
observed TU was determined from the relative inhibition value cor-
responding to the no-observed-effect concentration (NOEC) from the
mixture tests, with the calculated TU indicating the sum of the TUs
of individual pharmaceuticals based on their NOECs.
for the mixture were somewhat underestimated compared with
those predicted [27].
In the 21-d chronic mixture tests, the inhibited reproduction
rate was used as the endpoint, which can be determined by
the ratio of the number of neonates in various mixture series
of the three pharmaceuticals to those in the control. Figure 3
shows the expected and observed toxicities toward D. magna
of the diclofenac, ibuprofen, and clofibric acid mixtures. The
present results illustrate that the measured chronic toxicities
for the mixtures were greater than we predicted. In statistical
analysis, the observed TU had high standard deviations over
all the ranges tested. Overall, the measured toxicities of the
mixtures of the three pharmaceuticals in the present study were
not significantly different from the predicted toxicities for ei-
ther the acute or chronic toxicities, although a twofold increase
in the combined effect of PhACs on D. magna was observed.
Note, however, that the result of the mixture toxicity could be
serious if the combined toxic effects of unknown metabolized
chemicals and large number of PhACs in real water environ-
ments are considered.
Grab samples of the influents and effluents at five WWTPs
in metropolitan cities in South Korea were collected between
January and November of 2004 (Table 3). Recovery of the
pharmaceuticals ranged from 72 to 89%, with standard devi-
ations (1s, n 5 3) between 5 and 20%, following spiking of
the water with 1 mg/L of each compound. The present results
showed that residual concentrations of seven PhACs were de-
tected at concentrations ranging from 0.01 to 88.99 mg/L in
the influents and from 0.01 to 10.96 mg/L in the effluents.
Salicylic acid, ranging between 0.12 and 88.99 mg/L, had the
highest detected concentrations in the present study. Gemfi-
brozil appeared at nonquantifiable levels in the multiple sam-
ples from all sampling sites because of the low limit of de-
tection. The results in Table 3 also show highly fluctuating
removal rates, between 8.7 to 91.3%, indicating that the phar-
maceuticals in WWTPs may not be eliminated effectively by
the use of traditional treatment processes (i.e., activated sludge,
coagulation, flocculation, and sedimentation) at WWTPs [4].
Low removal rates were found for several PhACs in repeated
observations. Very low removal rates of 8.7 and 23.9% were
observed for acetaminophen and diclofenac, respectively, in
all sampling areas, whereas that for carbamazepine in the con-
ventional treatment system, which discharged into surface wa-
ters at a concentration as low as 0.16 mg/L, was 91.3%. This
was a notable result compared to that in another study [5],
which showed a very low removal rate (7%) for carbamazepine
in WWTPs. However, through analytical investigations on the
influents and effluents from different WWTPs in South Korea,
relatively high removal efficiencies were observed for most
pharmaceuticals, with the exception of acetaminophen, al-
though no notable differences were found between WWTPs
compared to results of previous surveys in other countries
[3,8]. This may have been caused by differences in the elim-
ination rates of the pharmaceutical residues because of sorption
and biodegradation. Meanwhile, treatment technologies, such
as oxidation with chlorine and ozone, activated carbon, and
membrane filtration, have been demonstrated as effective
methods for the elimination of antibiotics [29] and some phar-
maceuticals [21,30].
Figure 4 shows the effluent concentration profiles for seven
pharmaceuticals following treatment at the WWTPs in Korean
metropolitan cities (Gwangju1, Gwangju2, Busan, and Daegu).
Most residual PhAC concentrations were in the range 0.01 to
0.4 mg/L, including approximately 40% not-detectable points.

Table 3. Concentrations (mg/L) of selected pharmaceuticals in wastewater treatment plant influents and effluents from South Korea during the
2004 survey

Influent (mg/L) Effluent (mg/L)

REC 6 1sb Removal rate
Pharmaceuticals LOD a (%) Samples (n) Mean (max, min) n . LOD c Mean (max, min) n . LODc (%)

Diclofenac 0.01 85 6 7 17 2.59 (9.87, ND)d 14 1.97 (10.96, ND) 12 23.9

Ibuprofen 0.01 79 6 10 17 0.30 (0.58, ND) 14 0.07 (0.31, ND) 12 77.8
Clofibric acid 0.01 89 6 5 17 1.51 (4.38, 0.03) 17 0.31 (0.74, ND) 15 79.5
Carbamazepine 0.05 75 6 15 16 1.84 (9.42, ND) 9 0.16 (0.97, ND) 8 91.3
Salicylic acid 0.1 73 6 20 16 23.92 (88.99, 0.12) 15 2.43 (6.73, ND) 13 89.8
Gemfibrozil 0.5 83 6 9 16 ND (ND, ND) 0 ND (ND, ND) 0 ND
Acetaminophen 0.01 72 6 18 16 0.07 (0.26, ND) 14 0.06 (0.16, ND) 12 8.7
a LOD 5 limit of detection.
b Recoveries (REC) and standard deviation (1s, n 5 3) of pharmaceuticals, following spiking of the water with 1 mg/L of each compound.
c Number of samples having values greater than the LOD.
d ND 5 not detectable.
270 Environ. Toxicol. Chem. 25, 2006
Fig. 4. Comparative mean concentration (mg/L) profiles for the target
pharmaceuticals in the effluents from metropolitan cities (Gwangju,
Busan, and Daegu) in South Korea during 2004. An asterisk indicates
no detectable concentration. The data were less than the detection
limits in Seoul (South Korea).
However, several hundredfold higher concentrations were ob-
served for salicylic acid, up to 2.2 and 6.7 mg/L at Gwangju1
and Busan, respectively. These high concentrations in the ef-
fluents might affect ecosystems because of the large volumes
of water discharged daily into the nearby river. Interestingly,
none of the PhACs mentioned above were detected in the
effluents at the Seoul site (not shown in graph). Although only
one sample was taken from Seoul, the results were unexpected,
considering Seoul is the largest and most industrialized city
in Korea.
Many pharmaceuticals containing polar functional groups
are thermally stable and do not readily lend themselves to GC
analysis. Therefore, most GC analysis of polar pharmaceuticals
must incorporate a derivatization step to overcome this limi-
tation [30], which makes the sample preparation laborious and
time-consuming, increases the probability of contamination
and experimental errors, and leads to degradation of labile
compounds. In addition, pharmaceuticals are easily changed
to their metabolites by glucuronidation [31] or photodegra-
dation [32], although considerable amounts of their unchanged
forms are excreted via urine or feces. Therefore, further mea-
surements and identification of pharmaceuticals and their me-
tabolites in WWTP effluents and rivers will be required to
evaluate the contamination and occurrence of drug residues in
surface waters.
Risk assessment in the aquatic environment
The toxicity index (i.e., HQ) was calculated by comparing
the maximum measured concentrations of field data collected
from the WWTPs with the acute toxicity values to D. magna.
Figure 5 shows the effect of WWTP effluents that contained
pharmaceuticals was not a significant risk, even at the 95th
percentile contamination range, although a risk assessment fac-
tor of 1,000, as recommended by the European Union and the
Organization for Economic Co-operation and Development,
was applied. For salicylic acid and diclofenac, a difference of
less than one order of magnitude between maximum environ-
mental and effect concentrations was represented by the HQ
values. None of the HQ values of the drugs tested in the present
study exceeded 1. On average, the HQ values of the phar-
maceuticals measured in the WWTP effluents were within the
G.H. Han et al.
Fig. 5. Graphical illustration of the calculated hazard quotients (HQs)
for the selected pharmaceuticals in the wastewater treatment plants
effluents. A HQ of 1 indicates the critical value to determine a sig-
nificant risk of pharmaceuticals. A 5 acetaminophen; Carb 5 car-
bamazepine; Clof 5 clofibric acid; D 5 diclofenac; G 5 gemfibrozil;
I 5 ibuprofen; S 5 salicylic acid.
ranges of 1021 to 1024 when an application factor of 1,000 was
applied. From our results, it may be concluded that no risks
to D. magna were caused by the pharmaceuticals in Korean
WWTP effluents. Risk assessments also were conducted by
applying a factor of 100 for the chronic effect on the basis of
NOECs for the three pharmaceuticals (diclofenac, ibuprofen,
and clofibric acid). The present results indicated that the three
pharmaceuticals had no significant ecotoxicological impact on
long-term chronic exposure (not shown in graph). Moreover,
pharmaceuticals may be of less risk if the dilution, because of
the mixing of effluents and the receiving stream, is considered.
Many uncertainties, however, exist in risk assessments, es-
pecially those attributed to the difficulties in identifying the
presence of pharmaceuticals and their quantification in water
discharged from WWTPs containing a mixture of chemicals.
Currently, a method (i.e., extraction and derivatization) used
for the detection of pharmaceuticals in WWTP effluents with
lower detection limits is being developed. Metabolites can be
one of the concerns in the ecological impact of pharmaceu-
ticals; therefore, an analytical method for the detection of their
forms should be developed for more accurate risk assessments.
In addition, more toxicological data for various pharmaceu-
ticals must be obtained from bioassay experiments using aquat-
ic species, such as algae, daphnids, and fish. Sanderson et al.
[1] demonstrated that the susceptibility of aquatic species to
pharmaceuticals were in the following order: Algae, daphnids,
and fish. According to their results, approximately 10% of
pharmaceuticals in the environment had a HQ greater than 1
when multiplied by an application factor of 1,000. Conse-
quently, the potential risk of pharmaceuticals should be mon-
itored carefully, with more bioassay data, although no risk was
found in the WWTP effluents from large Korean cities.
CONCLUSION
The overall ecotoxicological effects of PhACs detected in
WWTP effluents were investigated by biological and chemical
analyses. The bioassay results showed LC50s and NOECs
ranging from levels of several ppm to several tens of ppm in
the acute and chronic tests, respectively, for the selected
PhACs. The combined toxicity of pharmaceuticals showed
Occurrence and toxicity of pharmaceuticals from Korean WWTPs
slight synergistic effects in both the acute and chronic tests.
These bioassay results would be valuable in risk assessments
with respect to pharmaceutical residues in surface waters, tak-
ing into account the insufficient toxicological data regarding
pharmaceuticals. The concentrations of PhACs in discharged
effluents indicated highly variable distributions depending on
the site, although relatively high removal efficiencies for most
pharmaceuticals (except for acetaminophen) were observed in
Korean WWTPs compared to those from other countries. The
PhAC residues in the effluents might affect aquatic ecosystems
because of the large volumes of water discharged daily into
the nearby river. Finally, the potential risk from the pharma-
ceuticals detected in the effluents from Korean WWTPs in-
dicated a low biological risk (HQ , 1) to aquatic ecosystems
when a risk assessment factor of 1,000 was applied to the tests
on D. magna. The monitored effluent concentrations did not
reach critically harmful levels. It was concluded that no sig-
nificant risk from the selected pharmaceuticals existed in Ko-
rean WWTPs, despite the presence of uncertainty that should
be characterized in the determination and toxicity of metab-
olites.
Acknowledgement—We acknowledge the financial support of the
Ministry of Environment as The Eco-technopia 21 project through
the Korea Institute of Environmental Science and Technology
(KIEST) and a grant (code 4-1-2) from the Sustainable Water Re-
sources Research Center of 21st Century Frontier Research Program.
We also thank Dong-il Shimadzu Corporation for technical and fi-
nancial support.
REFERENCES
1. Sanderson H, Johnson DJ, Wilson CJ, Brain RA, Solomon KR.
2003. Probabilistic hazard assessment of environmentally occur-
ring pharmaceuticals toxicity to fish, daphnids and algae by ECO-
SAR screening. Toxicol Lett 144:383–395.
2. Ferrari B, Paxéus N, Giudice RL, Pollio A, Garric J. 2003. Eco-
toxicological impact of pharmaceuticals found in treated waste-
waters: Study of carbamazepine, clofbric acid, and diclofenac.
Ecotoxicol Environ Saf 55:359–370.
3. Heberer T. 2002. Tracking persistent pharmaceutical residues
from municipal sewage to drinking water. J Hydrol 266:175–189.
4. Doll TE, Frimmel FH. 2003. Fate of pharmaceuticals photodeg-
radation by simulated solar UV-light. Chemosphere 52:1757–
1769.
5. Ternes TA. 1998. Occurrence of drugs in German sewage treat-
ment plants and rivers. Water Res 32:3245–3260.
6. Kolpin DW, Furlong ET, Meyer MT, Thurman EM, Zaugg SD,
Barber LB, Buxton HT. 2002. Pharmaceuticals, hormones, and
other organic wastewater contaminants in U.S. streams, 1999–
2000: A national reconnaissance. Environ Sci Technol 36:1202–
1211.
7. Buser HR, Poiger T, Muller MD. 1998. Occurrence and fate of
the pharmaceutical drug diclofenac in surface waters: Rapid pho-
todegradation in a lake. Environ Sci Technol 32:3449–3456.
8. Stumpf M, Ternes TA, Wilken RD, Rodrigues SV, Baumann W.
1999. Polar drug residues in sewage and natural waters in the
state of Rio de Janeiro, Brazil. Sci Total Environ 225:135–141.
9. Ahrer W, Scherwenk E, Buchberger W. 2001. Determination of
drug residues in water by the combination of liquid chromatog-
raphy or capillary electrophoresis with electrospray mass spec-
trometry. J Chromatogr A 919:69–78.
10. Öllers S, Singer HP, Fässler P, Müller SR. 2001. Simultaneous
quantification of neutral and acidic pharmaceuticals and pesticides
at the low-ng/L level in surface and waste water. J Chromatogr
A 911:225–234.
11. Stumpf M, Ternes TA, Haberer K, Baumann W. 1998. Isolierung
von ibuprofen-metaboliten and deren bedeutung als kontaminaten
in der aquatischen Umwelt. Vom Wasser 91:291–303.
12. Buser HR, Poiger T, Muller MD. 1999. Occurrence and environ-
Environ. Toxicol. Chem. 25, 2006 271
mental behavior of the pharmaceutical drug ibuprofen in surface
waters and in wastewater. Environ Sci Technol 33:2529–2535.
13. Kosjek T, Heath E, Krbavcic A. 2005. Determination of nonste-
roidal anti-inflammatory drug (NSAIDs) residues in water sam-
ples. Environ Int 31:679–685.
14. Sedlak DL, Pinkston KE. 2001. Factors affecting the concentra-
tions of pharmaceuticals released to the aquatic environment. Wa-
ter Resources Update 120:56–64.
15. Hirsch R, Ternes TA, Haberer K, Kratz KL. 1999. Occurrence of
antibiotics in the aquatic environment. Sci Total Environ 225:
109–118.
16. Ashton D, Hilton M, Thomas KV. 2004. Investigating the envi-
ronmental transport of human pharmaceuticals to streams in the
United Kingdom. Sci Total Environ 333:167–184.
17. Miao X-S, Metcalfe CD. 2003. Determination of carbamazepine
and its metabolites in aqueous samples using liquid chromatog-
raphy–electrospray tandem mass spectrometry. Anal Chem 75:
3731–3738.
18. Pedersen JA, Soliman M, Suffet IH. 2005. Human pharmaceu-
ticals, hormones, and personal care product ingredients in runoff
from agricultural fields irrigated with treated wastewater. J Agric
Food Chem 53:1625–1632.
19. Gross B, Montgomery-Brown J, Naumann A, Reinhard M. 2004.
Occurrence and fate of pharmaceuticals and alkylphenol ethox-
ylate metabolites in an effluent-dominated river and wetland. En-
viron Toxicol Chem 23:2074–2083.
20. Metcalfe CD, Koenig BG, Bennie DT, Servos M, Ternes TA,
Hirsch R. 2003. Occurrence of neutral and acidic drugs in three
effluents of Canadian sewage treatment plants. Environ Toxicol
Chem 22:2872–2880.
21. Ternes TA, Meisenheimer M, Mcdowell D, Brauch HJ, Brigitte
HG, Preuss G, William U, Zulei-Seibert N. 2002. Removal of
pharmaceuticals during drinking water treatment. Environ Sci
Technol 36:3855–3863.
22. U.S. Environmental Protection Agency. 1993. Method for mea-
suring the acute toxicity of effluents and receiving waters to fresh-
water and marine organism. EPA/600/4-90/027F. Cincinnati, OH.
23. U.S. Environmental Protection Agency. 1994. Short-term meth-
ods for estimating the chronic toxicity of effluents and receiving
waters to freshwater organisms. EPA/600/4-91/002. Cincinnati,
OH.
24. U.S. Environmental Protection Agency. 1992. Framework for
ecological risk assessment. EPA/630/R-92/001. Risk Assessment
Forum, Washington, DC.
25. Commission of the European Communities. 1996. Technical guid-
ance document in support of commission directive 93/67/EEC on
risk assessment for new notified substances and commission reg-
ulation (EC) No 1488/94 on risk assessment for existing sub-
stances. Office for Official Publications of the European Com-
munities, Luxembourg.
26. Organization for Economic Co-operation and Development. 1992.
Report of the OECD workshop on the extrapolation of laboratory
aquatic toxicity data to the real environment. Monograph 58.
Paris, France.
27. Cleuvers M. 2003. Aquatic ecotoxicity of pharmaceuticals in-
cluding the assessment of combination effects. Toxicol Lett 142:
185–195.
28. Halling-Sorensen B, Nielsen SN, Lanzky PF, Ingerslev F, Lutzhoft
HCH, Jorgensen SE. 1998. Occurrence, fate and effect of phar-
maceutical substances in the environment—A review. Chemo-
sphere 36:357–393.
29. Adams C, Wang Y, Loftin K, Meyer M. 2002. Removal of an-
tibiotics from surface and distilled water in conventional water
treatment processes. J Environ Eng 128:253–260.
30. Boyd GR, Reemtsma H, Grimm DA, Mitra S. 2003. Pharmaceu-
ticals and personal care products (PPCPs) in surface and treated
waters of Louisiana, USA, and Ontario, Canada. Sci Total En-
viron 311:135–149.
31. Richardson ML, Bowron JM. 1985. The fate of pharmaceutical
chemicals in the aquatic environment. J Pharm Pharmacol 37:
1–12.
32. Poiger TH, Buser HR, Muller MD. 2001. Photodegradation of
the pharmaceutical drug diclofenac in a lake: Pathway, field mea-
surement, and mathematical modeling. Environ Toxicol Chem 20:
256–263.
33. Howard PH, Meylan WM. 1997. Handbook of Physical Prop-
erties of Organic Chemicals. Lewis, Boca Raton, FL, USA.