Effluent From Drug Manufactures Contains Extremely High Levels of Pharmaceuticals

Abstract

It is generally accepted that the main route for human pharmaceuticals to the aquatic environment is via sewage treatment plants receiving wastewater from households and hospitals. We have analysed pharmaceuticals in the effluent from a wastewater treatment plant serving about 90 bulk drug manufacturers in Patancheru, near Hyderabad, India--a major production site of generic drugs for the world market. The samples contained by far the highest levels of pharmaceuticals reported in any effluent. The high levels of several broad-spectrum antibiotics raise concerns about resistance development. The concentration of the most abundant drug, ciprofloxacin (up to 31,000 microg/L) exceeds levels toxic to some bacteria by over 1000-fold. The results from the present study call for an increased focus on the potential release of active pharmaceutical ingredients from production facilities in different regions.

Full text

Journal of Hazardous Materials 148 (2007) 751–755
Short communication
Effluent from drug manufactures contains extremely
high levels of pharmaceuticals
D.G. Joakim Larsson a,∗, Cecilia de Pedro a, Nicklas Paxeusb
aInstitute of Neuroscience and Physiology, The Sahlgrenska Academy at G¨oteborg University, Box 434, SE-405 30 G¨oteborg, Sweden
bEnvironmental Chemistry, Gryaab AB, Norra F˚agelrov¨agen 3, SE-418 34 G¨oteborg, Sweden
Received 23 April 2007; received in revised form 29 June 2007; accepted 3 July 2007
Available online 6 July 2007
Abstract
It is generally accepted that the main route for human pharmaceuticals to the aquatic environment is via sewage treatment plants receiving
wastewater from households and hospitals. We have analysed pharmaceuticals in the effluent from a wastewater treatment plant serving about
90 bulk drug manufacturers in Patancheru, near Hyderabad, India—a major production site of generic drugs for the world market. The samples
contained by far the highest levels of pharmaceuticals reported in any effluent. The high levels of several broad-spectrum antibiotics raise concerns
about resistance development. The concentration of the most abundant drug, ciprofloxacin (up to 31,000 ␮g/L) exceeds levels toxic to some bacteria
by over 1000-fold. The results from the present study call for an increased focus on the potential release of active pharmaceutical ingredients from
production facilities in different regions.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Pharmaceuticals; Antibiotics; Environment; Effluent; Toxicity
1. Introduction
The release of pharmaceuticals from sewage effluents to
rivers and lakes is an issue of growing concern. Drugs are fre-
quently detected in effluents at levels from below 1 ng/L up to
afew␮g/L. Ethinylestradiol, the estrogen in many hormonal
contraceptives, is at least in part responsible for the feminiza-
tion of fish downstream from sewage treatment plants [1–3].
Propranolol [4], diclofenac [5], gemfibrozil [6], ibuprofen and
fluoxetine [7] are other examples of pharmaceuticals reported
to affect aquatic organisms at or around environmentally rele-
vant levels in laboratory experiments, but causal links between
the exposure to these drugs and any observed environmental
effects in the field have so far not been established. Antibiotic-
resistant bacteria are found in the aquatic environment, but to
what extent the antibiotics in the sewage effluents contribute to
this development is also not clear [8].
Current environmental risk assessment procedures in dif-
ferent regions focus on the release of active ingredients from
municipal sewage treatment plants [9,10]. Production facilities
∗Corresponding author. Tel.: +46 31 7863589; fax: +46 31 7863512.
E-mail address: joakim.larsson@fysiologi.gu.se (D.G.J. Larsson).
represent another potential way of entry of drugs to the environ-
ment [11]. The environmental standards of production facilities
is generally covered by a different set of regulations, although
without a similar focus on the potential release on active sub-
stances as for the registration and use of the final products [9,10].
Indeed, arguments have been raised that highly controlled pro-
duction processes, as well as the great value of the drugs, would
assure that only minor amounts of active substances would
escape [12]. Interestingly, there is to our best knowledge no
publicly available peer-reviewed information available that can
confirm or reject this claim. In this study we therefore hypoth-
esized that discharges of active ingredients during production
could be of substantial environmental concern. We began to
address this hypothesis by analysing active ingredients in a
common effluent from a large group of production facilities in
south-central India.
2. Materials and methods
2.1. Description of the treatment plant and sampling of
effluent
The investigated plant (Patancheru Enviro Tech Ltd.; PETL)
is situated in Patancheru, near Hyderabad. PETL receives
0304-3894/$ – see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.jhazmat.2007.07.008

752 D.G.J. Larsson et al. / Journal of Hazardous Materials 148 (2007) 751–755
approximately 1500 m3of wastewater per day, primarily from
about 90 bulk drug manufacturers. These industries comprise
examples of the entire production chain, via synthesis of inter-
mediates to active ingredients. The wastewater is transported
on trucks to PETL, where it is collected in a buffer cistern
with a retention time of approximately 2 days thereby ensur-
ing a less variable influent. After chemically assisted removal
of solids, about 20% raw domestic sewage is added to improve
biological treatment efficiency. The retention time in the aer-
ated/oxygenated biological treatment is about 4 days, followed
by settling in tanks and centrifugation of sludge. Some sludge is
fed back into the process. The content of organic material mea-
sured as BOD and COD is reported to be reduced from typically
1300 and 6000 mg/L, respectively in the mixed influent to 270
and 1400 mg/L in the treated effluent. Similarly, the amount of
total dissolved solids (TDS) and total suspended solids (TSS) is
reduced from about 9000 and 500 mg/L to 5000 and 300 mg/L,
respectively. The pH of both the influent and the treated effluent
is around 7.5. The estimated effluent volume of 1500 m3/day
is based on the reported incoming volumes and assuming that
the 20% added domestic sewage roughly equals the evaporation
and sludge removal. The clarified effluent is discharged in the
Isakavagu stream feeding the Nakkavagu, Manjira and eventu-
ally Godawari rivers. Solid waste is transported to a land fill unit.
With valuable assistance from local authorities and organi-
zations effluent was sampled from the treatment plant on two
consecutive days in November 2006 during normal operation
under the supervision of the Andhra Pradesh Pollution Con-
trol Board and PETL. The samples were frozen on dry ice and
shipped to Sweden for further analyses. The extensive mixing
of various influents (about 150 trucks per day) and long reten-
tion time in the plant (approximately 6.5 days) suggest that grab
samples will represent normal operation conditions reasonably
well.
2.2. Chemical analyses of pharmaceuticals in effluent
A contract lab (Analycen AB) first screened the samples
for the presence of 59 pharmaceuticals (Supplementary Table
S1). Based on these preliminary data we made a more precise
quantification of the nine most abundant drugs from the
screening plus two additional fluoroquinolones. The anal-
ysis was performed using Surveyor HPLC and LCQ-Duo
MS (ThermoFinnigan Inc., USA) acquiring MS/MS data
in ESI+ mode. The reference compounds (purity > 97% by
weight), LC–MS-grade solvents and other chemicals used for
analysis were purchased from Sigma–Aldrich Sweden AB
(Stockholm, Sweden), LGC Promochem AB (Bor˚
as, Sweden)
and Riedel-de Haen (Seelze, Germany). Chromatographic
separations were performed on two columns purchased from
Thermo Scientific (Waltham, MA, USA), namely Hypersil
Fluorophase RP (100 mm ×2.1 mm ID, packed with 5 ␮m
perfluorinated RP-C6; method 1; used for all fluoroquinolones
except ciprofloxacin) and Hypersil Gold (150 mm ×2.1 mm
ID, packed with 5 ␮m end-capped, base-deactivated RP-C18;
method 2; used for the rest of drugs and ciprofloxacin). In
both cases the column temperature was kept at 25 ◦C and the
flow rate at 200 ␮L min−1. Method 1: using solvents A (water,
0.1% formic acid) and B (methanol) the gradient program was
run as follows—isocratic 90% A and 10% B for 10 min, then
to 25% B in 5 min, then to 40% B in 15 min, then to 55% B
in 10 min, then to 70% B in 10 min and finally to 100% B in
5 min. Method 2: using solvents A (water, 15 mmol ammonium
formate) and B (acetonitrile) the gradient program was run as
follows—isocratic 96% A and 4% B for 8 min, then to 15%
B in 7 min, then to 55% B in 25 min and finally to 98% B in
5 min. MS/MS data were acquired in ESI+ mode (capillary
temperature 240 ◦C; sheath and auxiliary nitrogen gas flows
set to respectively 70 and 4; source voltage 4.50kV; source
current 80 ␮A; capillary voltage 29 V). The collision energy
required to produce the desired quantity of daughter ions was
individually optimized for each analyte. Detection by a selective
monitoring of daughter ions (parent ion MH+→daughter ions
monitored, with the underlined m/zbeing used for quantifi-
cation) is indicated as follows: cetirizin (389.1 →201.0),
citalopram (325.1 →262.1, 279.9 and 307.2), ciprofloxacin
(332.1 →288.2 and 314.2), enoxacin (321.1 →257.3,
277.2 and 303.2), enrofloxacin (360.1 →316.2, 245.1 and
217.1), lomefloxacin (352.1 →308.2, 288.3 and 265.2),
losartan (423.1 →207.2, 377.0 and 405.0), metoprolol
(268.2 →191.0 and 218.1), norfloxacin (320.1 →276.2 and
302.2), ofloxacin (362.1 →318.2 and 261.3) and ranitidine
(315.0 →270.0, 224.0 and 175.9). Although the concentrations
of the 11 analysed drugs were sufficiently high not to justify any
pre-concentration step, the removal of debris and particles in
order to protect the analytical equipment was found to be neces-
sary. Additionally, to diminish possible ion suppression effects
and assess correct quantification, a standard addition method
with parallel spiked samples and four-point (unknown plus
three spikes) calibration curve was used. Thus the pre-treatment
of the samples (native and spiked with known concentrations of
the pharmaceuticals) included acidification to pH 2 (phosphoric
acid), centrifugation at 13,500 rpm for 3 min (MiniSpin from
Eppendorf Nordic ApS, Hørsholm, Denmark) and filtration
(20 ␮m, glass-fibre filter from Millipore AB (Solna, Sweden))
before injection and analysis.
2.3. Toxicity tests
Standard toxicity tests were performed on thawed effluent
samples in Sweden. The acute effects on bioluminescence of the
bacteria Vibro fisheri were carried out using a Microtox M500
toxicity analyzer according to the manufacturer’s instructions
(Azur Environmental, Newark, Delaware, USA). Each sam-
ple/concentration was analysed in duplicate at 0, 1.25, 2.5, 5
and 10% dilutions. Immobilization of the water flea Daphnia
magna was performed according to EN ISO 6341:1996. Each
sample/concentration was analysed in quadruplicates (0, 0.6,
1.3, 2.5, 5, and 10%). Germination tests with salad seeds (Lac-
tuca sativa) were performed in Petri dishes according to [13] at
0, 1, 2, 5, 10, 20 and 50% dilutions using 120 seeds per con-
centration. After 5 days the number of seedlings penetrating
the cover sand was counted as well as the number of seedlings
developing cotyledons.

D.G.J. Larsson et al. / Journal of Hazardous Materials 148 (2007) 751–755 753
Table 1
Top 11 active pharmaceutical ingredients analysed in effluent samples from
PETL, a common effluent treatment plant near Hyderabad serving about 90
bulk drug manufacturers
Active ingredient Type of drug Range (␮g/L)
Ciprofloxacin Antibiotic-fluoroquinolone 28,000–31,000
Losartan Angiotensin II receptor antagonist 2,400–2,500
Cetirizine H1-receptor antagonist 1,300–1,400
Metoprolol ␤1-adrenoreceptor antagonist 800–950
Enrofloxacin Antibiotic-fluoroquinolone
(veterinary use)
780–900
Citalopram Serotonin reuptake inhibitor 770–840
Norfloxacin Antibiotic-fluoroquinolone 390–420
Lomefloxacin Antibiotic-fluoroquinolone 150–300
Enoxacin Antibiotic-fluoroquinolone 150–300
Ofloxacin Antibiotic-fluoroquinolone 150–160
Ranitidin H2-receptor antagonist 90–160
Drugs were analysed using LC–MS/MS monitoring at least two specific frag-
ment ions per substance when possible and quantified using a four-point
calibration. Data from two samples taken on consecutive days are presented.
3. Results and discussion
The initial screening of 59 pharmaceuticals suggested that 21
of these were present at concentrations above 1 ␮g/L (Table S1).
An independent, quantitative analysis in our laboratory of the
nine tentatively most abundant drugs and two additional antibi-
otics confirmed the findings of the screening. All 11 drugs were
detected at levels >100 ␮g/L (Table 1). To the best of our knowl-
edge, the concentrations of these 11 drugs were all above the
previously highest values reported in any sewage effluent.
We would like to highlight the exceptional concentrations
of fluoroquinolones found here, particularly ciprofloxacin—an
antibiotic produced by several companies in the area. Nor-
mally, all of the drugs, including ciprofloxacin, are found in
sewage effluents at concentrations around or below 1 ␮g/L
and occasionally at somewhat higher levels in discharges from
hospitals [14–20]. The concentrations of ciprofloxacin (up to
31,000 ␮g/L) were higher than the maximal therapeutic human
plasma levels. In an ecotoxicological context, the levels of
ciprofloxacin were orders of magnitude above the published
EC50 toxicity values for Microcystis aeruginosa (17 ␮g/L) and
Lemna minor (203 ␮g/L) [21]. The concentrations of lome-
floxacin, norfloxacin, ofloxacin, enrofloxacin and enoxacin also
exceed levels toxic to plants, diatoms, blue green algae and/or
other bacteria [21–24]. The discharge load of ciprofloxacin
corresponds to approximately 45 kg of active pharmaceutical
ingredient per day, which is equivalent to the total amount con-
sumed in Sweden (population nine million) over an average
5-day period [12].
Of further concern is that the industrial effluent is mixed with
human sewage within the plant to improve biological treatment
efficiency. Hence, there is a risk that pathogens will be exposed
to antibiotics for prolonged periods. Ciprofloxacin is genotoxic
and induces horizontal transfer of resistance between different
species of bacteria, effects that may be observed at concentra-
tions as low as 5–10 ␮g/L [14,15,25]. Therefore, the recipient
waters and the treatment plant itself may be spawning grounds
for resistant bacteria. One may also anticipate a reduced over-
all performance of the plant due to the expected toxicity of the
pharmaceuticals to the microorganisms within the plant. More-
over, the microbial flora downstream from the plant is likely to
be severely affected by the mixture of residual fluoroquinolones
[22]. Thus, there are multiple reasons to consider alternatives
to normal biological treatment for the removal of high levels of
antibiotic residues from wastewater.
In addition to several broad-spectrum antibiotics being
present, the list contained well-known drugs of different classes
of diverse chemical structure, frequently used to treat allergies,
ulcers, hypertension, migraine, depression and other common
disorders. For most of these non-antibiotic drugs, there is yet
insufficient chronic effects data on organisms likely to have
highly conserved target molecules with humans (i.e. fish) to
make adequate risk assessments. For example, citalopram is
known to affect the behaviour of fish [26] but the dose–response
relationship remains to be established. Citalopram has previ-
ously been reported in sewage effluents up to 612ng/L [27].
The amounts of pharmaceuticals detected could be expressed
in economical terms: if the equivalent amount of the 11 most
abundant active substances released during 24h were to be pur-
chased as final products in a Swedish pharmacy, they would cost
over D100,000 even if generic brands were selected (data not
shown). However, the production cost of the bulk drugs would
apparently be much lower than the price paid by the final con-
sumer. Since measures to minimize the release of certain drugs
during production may require significant investments, a high
value of the final product does not necessarily guarantee that
only trace amounts would be present in the waste [12].
A limitation of the study is that samples were collected on 2
days only. It is quite possible that the highest concentrations
found reflect individual deliveries of waste to the treatment
plant containing extreme quantities of drugs. However, the high
concentrations of active ingredients for many different types
of drugs strongly suggest that several industries are contribut-
ing. Thus, deliveries with high contents of drugs to PETL are
not isolated, unique events. The addition of raw sewage (20%)
to the process has likely contributed with some pharmaceuti-
cal residues but these amounts would be low compared to the
highest values analysed here.
Despite the fact that the applied toxicity tests may be con-
sidered crude, they were sufficiently sensitive to identify high
toxicities of the effluent to different types of organisms (Table 2).
Table 2
Toxicities of effluent samples from PETL sampled on two consecutive days in
November 2006
Test organism Duration
of test
Endpoint EC50
range (%)
Vibrio fisheri 15 min Luminescence 3
Daphnia magna 48 h Immobility 6.7–7.2
Lactuca sativa 120 h Emerging seedlings 17–35
L. sativa 120 h Developed cotyledons 1.6–3.2
Data are expressed as EC50-values, i.e. the concentrations of effluent required to
reduce the measured endpoint to 50% (Vibrio,Lactuca) or to immobilize 50%
of the Daphnia.

754 D.G.J. Larsson et al. / Journal of Hazardous Materials 148 (2007) 751–755
The toxicity to L. sativa is in agreement with reports that irri-
gated fields in Patancheru are no longer productive [28]. Based
on these toxicity tests alone, it is not possible to assign the
toxicity to a particular drug(s) or other constituents of the efflu-
ent. For example, the fluoroquinolone antibiotics, including
ciprofloxacin, are not very potent in the short-term Microtox
test despite that it is a bacterial toxicity test [29]. Nevertheless,
from the chemical analyses together with published toxicity data
it is undisputable that the levels of fluoroquinolones found in
the effluent are very toxic. It should be stressed that most drugs
have human target proteins. Other tests could reveal even higher
toxicities than the tests applied in this study.
The treated effluent studied does not constitute the only, or
even worst, contribution to environmental pollution by local
industries in Patancheru. Dumping of untreated industrial waste
is a recognized problem [30]. There are reports in the non-peer-
reviewed literature on severe environmental problems in nearby
villages, including deaths of cattle as well as a number of human
health issues [28]. This urgently motivates a thorough search for
responsible causes and appropriate mitigation measures. Drugs
produced in Patancheru are to a large extent globally distributed
and incorporated into products marketed by other pharmaceu-
tical companies. This implies that the environmental impact of
the production is not only a matter of local concern.
The present study demonstrates that there are production
facilities that release substantial amounts of drugs to the aquatic
environment. To the best of our knowledge such information has
previously not been reported in the peer-reviewed literature and
at present it is not possible to say how widespread the problem is.
It is plausible that the overall amount of drugs reaching the envi-
ronment via excretion from humans and via incorrect disposal
is larger than the amount released from production facilities on
the global scale. However, the present study demonstrates that
production facilities may be the most important point sources
in specific locations and the source for the highest environmen-
tal concentrations. The data presented here call for extended
investigations on the effluent quality, including the release of
active pharmaceutical ingredients, from production facilities in
different regions of the world.
Acknowledgements
We thank Ann-Sofie Wernersson, Marie Adamsson, the
Andhra Pradesh Pollution Control Board and Gamana for input
and assistance with sampling, Patrik Karlsson (Lantm¨
annen
AnalyCen AB) for the initial screening and SIDA, MISTRA,
FORMAS and the Swedish Research Council for financial
support.
Appendix A. Supplementary data
Supplementary data associated with this article can be found,
in the online version, at doi:10.1016/j.jhazmat.2007.07.008.
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