Ecotoxicology and Environmental Safety 136 (2017) 31–39

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Ecotoxicology and Environmental Safety

journal homepage: www.elsevier.com/locate/ecoenv

Ecological risk assessment of pharmaceuticals in the receiving environment

of pharmaceutical wastewater in Pakistan
crossmark
⁎
Muhammad Ashfaqa,b, , Khujasta Nawaz Khana, Muhammad Saif Ur Rehmanc,
Ghulam Mustafaa, Muhammad Faizan Nazara, Qian Sunb, Javed Iqbalc, Sikandar.I. Mullab,
Chang-Ping Yub
a
Department of Chemistry, University of Gujrat, H. H. Campus, Gujrat 50700, Pakistan
b
Institute of Urban Environment, Chinese Academy of Sciences, Xiamen 361021, China
c
Department of Chemical Engineering, COMSATS Institute of Information Technology, Lahore 54000, Pakistan

A R T I C L E I N F O A BS T RAC T

Keywords: The pharmaceutical industry of Pakistan is growing with an annual growth rate of 10%. Besides this growth, this
Pharmaceutical compounds industry is not complying with environmental standards, and discharging its effluent into domestic wastewater
Formulation units network. Only limited information is available about the occurrence of pharmaceutical compounds (PCs) in the
HPLC-UV environmental matrices of Pakistan that has motivated us to aim at the occurrence and ecological risk
Liquid-liquid extraction
assessment of 11 PCs of different therapeutic classes in the wastewater of pharmaceutical industry and in its
Risk assessment
receiving environmental matrices such as sludge, solid waste and soil samples near the pharmaceutical
formulation units along Shiekhupura road, Lahore, Pakistan. Target PCs (paracetamol, naproxen, diclofenac,
ibuprofen, amlodipine, rosuvastatin, ofloxacin, ciprofloxacin, moxifloxacin, sparfloxacin and gemifloxacin) were
quantified using in-house developed HPLC-UV. Ibuprofen (1673 µg/L, 6046 µg/kg, 1229 µg/kg and 610 µg/kg),
diclofenac (836 µg/L, 4968 µg/kg, 6632 µg/kg and 257 µg/kg) and naproxen (464 µg/L, 7273 µg/kg, 4819 µg/
kg and 199 µg/kg) showed the highest concentrations among 11 target PCs in wastewater, sludge, solid waste
and soil samples, respectively. Ecological risk assessment, in terms of risk quotient (RQ), was also carried out
based on the maximum measured concentration of PCs in wastewater. The maximum RQ values obtained were
with paracetamol (64 against daphnia), naproxen (177 against fish), diclofenac (12,600 against Oncorhynchus
mykiss), ibuprofen (167,300 against Oryzias latipes), ofloxacin (81,000 against Pseudomonas putida) and
ciprofloxacin (440 against Microcystis aeruginosa). These results show a high level of ecological risk due to the
discharge of untreated wastewater from pharmaceutical units. This risk may further lead to food web
contamination and drug resistance in pathogens. Thus, further studies are needed to detect the PCs in crops
as well as the government should strictly enforce environmental legislation on these pharmaceutical units.

1. Introduction manufacturing facilities, hospitals, agricultural and land runoff, house-

Rapid growth in human population, urbanization and industrializa-
tion has caused a proportional increase in the environmental contam-
ination with several inorganic and organic pollutants (Belhaj et al.,
2015). Among these pollutants, pharmaceuticals are considered as
emerging contaminants due to their frequent use, detection in different
environmental matrices in high concentrations and the potential risk to
the ecosystem (Verlicchi et al., 2012a, 2012b; Lombardo-Agui et al.,
2014). Pharmaceuticals are complex molecules, mostly organic in
nature with different physicochemical and therapeutic properties
(Kummerer, 2009). These pharmaceutical compounds (PCs) ultimately
become the part of the environment via various point sources such as
hold use and improper disposal. It is generally assumed that the release
of PCs from pharmaceutical production plants is negligible
(Kummerer, 2009). Usually, these production facilities employ good
manufacturing practices (GMPs), comply with environmental legisla-
tions, and they try to recover active pharmaceutical ingredients
(Kummerer, 2009). However, this hypothesis does not exist valid as
researchers have detected high concentrations of PCs in industrial
wastewater (Cardoso et al., 2014). It is therefore necessary to expand
the investigation regarding the occurrence, distribution and risk
assessment of PCs (Birch et al., 2015; Hughes et al., 2013) particularly
in highly populated developing countries.
In our previous study, we have discussed the occurrence and

⁎
Corresponding author at: Department of Chemistry, University of Gujrat, H. H. Campus, Gujrat 50700, Pakistan.
E-mail addresses: ashfaqchemist@hotmail.com, ashfaq@iue.ac.cn (M. Ashfaq).

http://dx.doi.org/10.1016/j.ecoenv.2016.10.029
Received 17 March 2016; Received in revised form 18 September 2016; Accepted 23 October 2016
Available online 01 November 2016
0147-6513/ © 2016 Elsevier Inc. All rights reserved.
M. Ashfaq et al. Ecotoxicology and Environmental Safety 136 (2017) 31–39

Fig. 1. a) Chemical structures of selected pharmaceuticals b) Location of sampling point of different types of samples. X, D, E, P and Y represent different sampling sites whereas the
symbols in yellow represents wastewater samples, green for sludge samples, red for solid waste samples and blue for soil samples.

32
M. Ashfaq et al.
distribution of PCs in the environmental matrices of developing
countries such as Bangladesh, China, India and Pakistan (Rehman
et al., 2015). We have established that the relocation of global
pharmaceutical industry in these countries and their non-compliance
with environmental legislation has led to the release of untreated
pharmaceutical wastewater into domestic wastewater network that
may develop drug resistant microbes. Recent studies have reported
that pharmaceutical industries in Pakistan do not comply with waste-
water treatment practices and discharge their untreated wastewater
into sewage. Such practice has not only witnessed higher concentration
of antibiotics and drugs into industrial wastewater, irrigated soil, plants
and groundwater (Khan et al., 2013; Hussain et al., 2016a, 2016b), but
it has also led to the detection of antibiotic resistance genes in the
receiving environment of pharmaceutical wastewater (Khan et al.,
2013). Still, the occurrence, distribution and accumulation of PCs in
different environmental matrices is not well studied (Du et al., 2014;
Ginebreda et al., 2010; Schaider et al., 2014) particularly in developing
countries (Carmona et al., 2014; Kookana et al. 2014). Kookana et al.
(2014) have emphasized the broad monitoring of PCs in the environ-
mental matrices of low income Asian countries in order to document
baseline data for their environmental concentration and relevant
ecological foot printing of these PCs.
Pharmaceutical industry is one of the main sectors in Pakistan
growing at the rate of 10% per annum. This sector exports variety of
PCs to more than 27 countries around the world. There are more than
386 pharmaceutical units of varying capacity. Besides such promising
economic indicators, local pharmaceutical industry does not comply
with National Environmental Quality Standards (NEQS). It releases its
wastewater into domestic wastewater networks without any pretreat-
ment (Rehman et al., 2015). Resultantly, pharma industry can cause
the excessive release of PCs into the natural environment as found in
other countries (Li et al., 2008; Lin and Tsai, 2009; Lin et al., 2008; Cui
et al., 2006; Fick et al., 2009; Larsson et al., 2007; Cardoso et al., 2014;
Phillips et al., 2010; Prasse et al., 2010; Sim et al., 2011). Besides this
fact, only limited data is available about the occurrence of PCs in the
environment of Pakistan whereas, the risk assessment is almost scarce.
Risk assessment of PCs in the environment is needed to know the
ultimate effect of PCs on the ecosystem. Verlicchi and coworkers have
carried out several risk assessment studies for PCs in the environ-
mental matrices (Verlicchi et al., 2012a, 2012b, 2014; Verlicchi and
Zambello, 2014, 2015).
The discharged PCs can cause adverse effects to the ecosystem of
receiving environmental matrices (wastewater, drinking water, surface
water, sediment and soil). These PCs may pose the risk of ecotoxicology
in many organisms including fish in the aquatic ecosystem (Fent et al.,
2006) and may also interfere with hormone synthesis or reproduction
(Mimeault et al., 2005; Nash et al., 2004; Sanchez et al., 2011). In case
of antibiotics, it is generally accepted that increased exposure of
microbial communities in the environment can result in the generation
and spread of antibiotic resistance genes in human pathogens
(Rutgersson et al., 2014; Forslund et al., 2013; Wellington et al.,
2013). Recent studies have brought the challenge of antibiotic resis-
tance to the highest level speculating a post antibiotic era ahead
(Teillant et al., 2015; Liu et al., 2015). Thus, it has become very much
important to investigate the occurrence and distribution of PCs,
released from pharmaceutical industrial wastewater, in the receiving
environmental matrices of Pakistan. It is equally important to carry out
the risk assessment of these PCs so that there ecotoxicology potential
must be known. This effort will provide baseline data that will lay the
foundations for further such investigations.
The aim of the present study was therefore the detection of some
commonly formulated and used pharmaceuticals in different environ-
mental matrices near pharmaceutical industrial area of Lahore-
Sheikhupura road. The industrial area is housing many pharmaceutical
formulation units along with residential area. The different pharma-
ceuticals were detected in wastewater, sludge, solid waste and soil
33
Ecotoxicology and Environmental Safety 136 (2017) 31–39
samples collected from catchment areas. In addition, ecological risk
assessment was conducted against different aquatic species. No such
studies have been reported for the detection of pharmaceuticals in
Pakistan. Therefore the results of this study will initiate an effort to
continue such type of studies in Pakistan.
2. Materials and methods
2.1. Chemicals and reagents
Reference standards of all the pharmaceutical compounds were
supplied by Schazoo Laboratories Pvt. Ltd. and Irza Pharma Pvt. Ltd.,
Lahore, Pakistan. The structures of these pharmaceuticals are given in
Fig. 1a. The rest of chemicals used were either HPLC grade or
analytical reagent grade. These chemicals were used without further
purification. All the solutions were filtered through 0.45 µm membrane
filters (Millipore, USA) before use.
2.2. Sampling
Wastewater, sludge and solid waste samples were collected from
suitable locations near pharmaceutical industrial units located around
Dosaco Chowk, 9–10 km Sheikhupura Road, Lahore-Pakistan. Solid
waste sample included the dust collected after the cleaning of equip-
ment. The soil samples were collected from nearby fields that were
irrigated with the pharmaceutical industry wastewater. The sampling
area was comprised of about 10 pharmaceutical formulation units.
These units formulate variety of PCs in bulk quantities. Five sampling
points were selected for the collection of wastewater, sludge and solid
waste samples (in the front and along the pharmaceutical units). The
soil samples (three different sampling points) were collected from the
nearby agriculture fields which were irrigated with pharmaceutical
wastewater. The sampling campaign was carried out (at different
timings in the same day) during the month of April 2014. Five samples
of each matrix (wastewater, sludge and solid waste), and three samples
of soil were collected in the morning. The same sampling campaign was
repeated in the afternoon. Two samples from each sampling point
(morning and afternoon) were later properly mixed to make one
composite sample for each site. These composite samples were
analyzed independently to ensure the quality of analyses, and to
account for any sampling uncertainty. The results have been reported
as mean ± SD of five composite samples for wastewater, sludge and
solid waste whereas, three composite samples for soil.
Sludge, solid waste and soil samples were collected in plastic bags
and were dried before extraction. One liter of all the wastewater
samples was collected in amber glass bottles. Immediately after sample
collection, each collected wastewater sample was added with about
250 mg of disodium edetate to complex the metal ions and, was then
stored in refrigerator at 4 °C to inhibit bacterial growth. The samples
were extracted within 48 h of sample collection. The sampling map is
given in Fig. 1b. Sampling points (X, D, E, P and Y) for wastewater,
sludge and solid waste samples have been shown in oval shape.
2.3. Extraction and chromatographic conditions
The targeted PCs were extracted from wastewater, sludge, solid
waste and soil samples employing liquid-liquid extraction. Chloroform
was selected as the final extractant among the initially chosen di-ethyl
ether, n-hexane and chloroform based on our previous experience
(Ashfaq et al., 2016a, 2016b). Liquid samples were first filtered using
0.45 µm membrane filters. Each filtered sample was extracted three
times using 50 mL chloroform for each extraction. The extracts were
combined, and evaporated. The residue was dissolved in 2 mL of
methanol and then, it was refrigerated till analysis. For the extraction
of PCs from solid matter (solid waste, sludge and soil samples), 10 g of
each matter was extracted three times with chloroform, filtered,
M. Ashfaq et al. Ecotoxicology and Environmental Safety 136 (2017) 31–39

evaporated, dissolved in 2 mL of methanol and then refrigerated till

further analyses.
For the analysis of targeted compounds, LC-20A system (Shimadzu,
Japan) equipped with UV detector was used. The samples were injected
using auto sampler with 20 µL loop. Two different validated HPLC
methods were used for the detection of all the eleven targeted
compounds. The first method was used to quantify paracetamol,
naproxen, diclofenac, ibuprofen, amlodipine and rosuvastatin (Group
1 PCs). The second method was used for the quantification of ofloxacin,
ciprofloxacin, sparfloxacin, moxifloxacin and gemifloxacin (Group 2
PCs). These methods can be found elsewhere in the literature (Ashfaq
et al., 2016a, 2016b). In brief, discovery C18 column (250×4.6 mm,
5 µm particle size) was used as stationary phase for both the methods
at room temperature. The mobile phase for Group 1 PCs consisted of a
mixture of acetonitrile and 0.1 M ammonium acetate (pH 5.0) in
50:50 v/v ratio. For Group 2 PCs, the mobile phase was a mixture of
methanol and 0.02 M phosphate buffer (pH 3.9) in the ratio of 40:60
(v/v). Phosphate buffer (pH 3.9) was prepared by adding 1 mL
triethylamine in 1 L 0.02 M potassium dihydrogen phosphate and its
pH was adjusted with o-phosphoric acid. The detection was carried out
at 254 nm for Group 1 PCs and at 279 nm for Group 2 PCs. All the
experiments were performed isocratically at 1 mL/min flow rate of the
mobile phase. The targeted analytes were quantified using external
standard method. The retention characteristics, quantification limits,
correlation and recovery of all analytes are given in Table 1.

2.4. Risk assessment

The impact of pharmaceutical pollution on the aquatic environment

was assessed through risk assessment measurements. The guidelines
issued by the European Agency for Evaluation of Medicinal Products
were used to calculate ecological risk assessment (EMA, 2006). The risk
assessment is generally expressed in terms of risk quotient (RQ) or
hazard quotient using the following equation: Where RQ represents
risk quotient or hazard quotient, MEC is the maximum measured
environmental concentration in µg/L and PNEC is the predicted no
effect concentration (µg/L). Value of RQ > 1 indicates high risk to the
aquatic community whereas RQ < 1 indicates medium or no risk. MEC
values were calculated using this study whereas; PNEC values were Fig. 2. a) Detection Frequency (DF) of PCs in various environmental matrices (DF has
taken from the literature. been calculated by dividing the number of detected samples by the number of total
samples) b) Concentration of pharmaceuticals in wastewater samples. The error bar in
the box and whisker diagram showing minimum to maximum values..
3. Results and discussions
mestic wastewater network without any treatment. We have focused to
The pharmaceutical industrial sector of Pakistan is considerably
assess the occurrence of 11 frequently formulated drugs in the different
growing due to increase in local population as well as export oppor-
environmental matrices near pharmaceutical formulation units of
tunities. However, the pharmaceutical industry does not comply with
Shiekhupura, Lahore-Pakistan. The occurrence of these drugs in
any local as well as international environmental standards. The
various environmental matrices is presented in Figs. 2–5. Most of
pharmaceutical industrial wastewater is directly discharged into do-
the targeted PCs were readily detected in all of the matrices. However,
the concentration of different PCs did vary with the sampling location.
Table 1
Retention characteristics, linearity, LOD/MQL, correlation and recovery of analytes.
The detection frequency of target PCs is given in Fig. 2a.

Analyte Retention time LOQ MQL Correlation Recovery (%) 3.1. Occurrence of PCs in wastewater
(min) (µg/L) (µg/L)

Paracetamol 2.8 30 3 0.988 73.4 Ten PCs were detected in wastewater samples collected from
Rosuvastatin 3.5 40 4 0.998 72.2 various points except gemifloxacin as shown in Fig. 2a. The concentra-
Amlodipine 4.2 80 8 0.989 72.9 tion of these PCs ranged from Below detection limit (BDL)-1673 µg/L
Naproxen 4.5 80 8 0.989 71.6 as shown in Fig. 2b and Table 2. Ibuprofen occurred at the highest
Diclofenac 5.5 120 12 0.999 70.9
Ibuprofen 9.7 200 20 0.999 72.0
concentration in wastewater (703–1673 µg/L) followed by diclofenac
Ofloxacin 5.6 25 0.5 0.999 79.9 (252–836 µg/L) and naproxen (215–464 µg/L). Ofloxacin (1.2–81 µg/
Ciprofloxacin 6.1 5.0 0.1 0.999 79.7 L), paracetamol (12–64 µg/L), rosuvastatin (15–61 µg/L) and amlodi-
Sparfloxacin 10.7 25 0.5 0.989 81.3 pine (14–51 µg/L) were detected in the medium concentration range
Moxifloxacin 11.8 33 0.66 0.998 83.7
(1.2–81 µg/L). Moxifloxacin (0.7–0.9 µg/L), ciprofloxacin (BDL-
Gemifloxacin 18.6 1.6 0.032 0.998 76.6
2.2 µg/L), and sparfloxacin (17–19 µg/L) were detected in the lowest
LOQ: Limit of quantification. concentration range of BDL-19 µg/L. The possible reason for the
MQL: Method quantification limit. absence of gemifloxacin might be due to its less production and use.

34
M. Ashfaq et al. Ecotoxicology and Environmental Safety 136 (2017) 31–39

Table 2
Concentrations of pharmaceuticals in different wastewater samples.

Concentration detected (µg/L)

Site 1 Site 2 Site 3 Site 4 Site 5

Paracetamol BDL 64 12 BDL 41

Naproxen BDL 270 215 451 464
Diclofenac 836 BDL 252 BDL BDL
Ibuprofen BDL 703 BDL 1673 900
Amlodipine 19 14 BDL 51 BDL
Rosuvastatin 20 61 18 25 15
Ofloxacin 81 BDL 1.2 BDL 50
Ciprofloxacin BDL BDL BDL 2.2 BDL
Sparfloxacin BDL BDL 19 BDL 17
Moxifloxacin 0.7 0.9 BDL BDL BDL
Gemifloxacin BDL BDL BDL BDL BDL

Fig. 3. Concentration of pharmaceuticals in sludge samples. The error bar in the box and
whisker diagram showing minimum to maximum values.
Fig. 4. Concentration of pharmaceuticals in solid waste samples. The error bar in the
box and whisker diagram showing minimum to maximum values.
Fig. 5. Concentration of pharmaceuticals in soil samples. The error bar in the box and
whisker diagram showing minimum to maximum values.
Fig. 2a showed that rosuvastatin and naproxen were the most abundant
PCs in wastewater samples with the higher detection frequency (80–
100%). Moreover, paracetamol, ibuprofen, amlodipine and ofloxacin
were detected with an average detection frequency (60%). Diclofenac,
moxifloxacin, sparfloxacin and ciprofloxacin were detected with a
below average detection frequency (20–40%) whereas, gemifloxacin
could not be detected at all. These results clearly showed that PCs of
Group 1 (average detection frequency=67%) were present abundantly
in wastewater compared with PCs of Group 2 (average detection
frequency=32%).
The concentration of the different PCs detected in this study is quite
high compared to their concentration reported in case of other
countries. Guerra et al. (2014) have reported the maximum concentra-
tion of ibuprofen (45 µg/L) and naproxen (250 µg/L) in wastewater of
Canada. Agunbiade and Moodley (2016) reported the concentrations of
ibuprofen (1.38 µg/L) and diclofenac (22.27 µg/L) in wastewater
samples of South Africa. Vergeynst et al. (2015) detected various PCs
in wastewater of Lede, Belgium, and reported the concentrations of
ibuprofen (5.7 µg/L), ciprofloxacin (0.278 µg/L), diclofenac (0.507 µg/
L), moxifloxacin (0.149 µg/L), naproxen (4.1 µg/L) and paracetamol
(67.1 µg/L). Camacho-Muñoz et al. (2014) characterized industrial
wastewater in Seville, Spain and reported the concentrations of
ibuprofen (3.11 µg/L), diclofenac (0.14 µg/L), ciprofloxacin (0.23 µg/
L), naproxen (2.34 µg/L) and ofloxacin (2.38 µg/L). This literature
comparison reveals that the concentrations of target PCs detected in
other countries is almost several hundred times less than our reported
concentrations. This difference in concentrations exists due to abso-
lutely no wastewater treatment in Pakistan pharmaceutical industry.
The most comparable study with our investigation was carried out
by Larsson et al. (2007) in Hyderabad, India. They analyzed the
effluents of a wastewater treatment plant receiving wastewater from
around 90 bulk drug manufacturers. The effluent samples contained
ciprofloxacin up to 31,000 µg/L and ofloxacin up to 160 µg/L. Fick
et al. (2009) investigated the same area and reported the concentration
of ciprofloxacin (14,000 µg/L) and ofloxacin (55 µg/L). Two recent
studies carried out in other industrial estates of Lahore also support
our results (Hussain et al., 2016a, 2016b; Khan et al., 2013). Hussain
et al. (2016a) monitored the occurrence of antibiotics in different
receiving environmental matrices of pharmaceutical effluent dis-
charged from localized industrial sectors of Lahore, Pakistan. The
results showed that ciprofloxacin (5250 µg/L) and ofloxacin (4120 µg/
L) were detected in pharmaceutical industrial wastewater. These
concentrations seem higher than our results because our sampling
area consists of about only 10 pharmaceutical units as compared to the
other studies (Larsson et al., 2007; Hussain et al., 2016a). Khan et al.
(2013) studied the occurrence of antibiotics in the proximity of our
study area and reported ciprofloxacin (6.2 µg/L) and ofloxacin (7.3 µg/
L) than our results Ofloxacin (1.2–81 µg/L) and ciprofloxacin (BDL-
2.2 µg/L) in pharmaceutical wastewater. Similarly, Terzic et al. (2008)
analyzed the pharmaceutical industrial wastewater in Bosnia Sarajevo
(Bosnia and Herzegovina) and Galenika in Belgrade (Serbia). Only
ibuprofen (420 µg/L) concentration was comparable to our results and
the concentration of other PCs was well below than the values observed
in our study. Sim et al. (2011) reported the concentrations of
paracetamol (104 µg/L), ciprofloxacin (34.6 µg/L), diclofenac
(203 µg/L) and naproxen (206 µg/L) in the effluent from pharmaceu-
tical production plant in South Korea (Sim et al., 2011). Lin et al.

35
M. Ashfaq et al.
(2008) detected different pharmaceutical compounds near pharmaceu-
tical production facilities in Taiwan and observed ciprofloxacin (9.6 µg/
L), ofloxacin (13.6 µg/L), ibuprofen (14.5 µg/L), naproxen (8.5 µg/L)
and diclofenac (29.8 µg/L) in wastewater. The reported concentrations
of PCs are significantly lower than the concentrations reported in our
study.
3.2. Occurrence of PCs in sludge
Nine target PCs were detected in the sludge samples collected from
various points except sparfloxacin and gemifloxacin as shown in
Fig. 2a. The concentration of these PCs ranged from BDL-7273 µg/kg
as presented in Fig. 3. Naproxen occurred at the highest concentration
in sludge (854–7273 µg/kg) followed by ibuprofen (2053–6046 µg/kg)
and diclofenac (695–4968 µg/kg). Paracetamol (BDL-483 µg/kg), ci-
profloxacin (BDL-454 µg/kg), amlodipine (153–372 µg/kg), ofloxacin
(132–347 µg/kg) and rosuvastatin (67–290 µg/kg) were detected in
the medium concentration range (BDL-483 µg/kg). Moxifloxacin was
detected in the lowest concentration range of BDL-235 µg/kg. Fig. 2a
showed that amlodipine, diclofenac, ibuprofen, naproxen and rosuvas-
tatin were the most abundant PCs in the sludge samples with 100%
detection frequency. Ciprofloxacin, ofloxacin, moxifloxacin and para-
cetamol were detected at few sampling sites with an average detection
frequency (40–60%). Gemifloxacin and sparfloxacin could not be
detected at any of the sampling sites. These results clearly showed
that PCs of Group 1 (average detection frequency ≥90%) were present
dominantly in the sludge samples compared with PCs of Group 2
(average detection frequency=28%).
The concentration of the different PCs detected in this study is quite
high compared to their concentration reported in case of other
countries. Taylor-Smith (2015) has reviewed the literature and re-
ported that much higher concentrations of ciprofloxacin (48,000 µg/
kg), ofloxacin (58,000 µg/kg), diclofenac (424 µg/kg), ibuprofen
(548 µg/kg) and naproxen (242 µg/kg) had been detected in sewage
sludge. Agunbiade and Moodley (2016) reported the concentration of
ibuprofen (0.009 µg/kg), ciprofloxacin (0.006 µg/kg) and diclofenac
(0.22 µg/kg) in the sludge samples of South Africa. Matongo et al.
(2015) also reported the concentration of ibuprofen (0.009 µg/kg),
ciprofloxacin (0.006 µg/kg) and diclofenac (0.22 µg/kg) in the sludge
samples of South Africa.
3.3. Occurrence of PCs in solid waste
All of the target PCs were detected in the solid waste samples
collected from various points as shown in Fig. 2a. The concentration of
these PCs ranged from BDL-6632 µg/kg as presented in Fig. 4.
Diclofenac occurred at the highest concentration in the solid waste
samples (125–6632 µg/kg) followed by naproxen (2018–4819 µg/kg)
and ibuprofen (133–1229 µg/kg). Paracetamol (BDL-874 µg/kg), ci-
profloxacin (2.1–161 µg/kg), ofloxacin (BDL-127 µg/kg), moxifloxacin
(BDL-106 µg/kg) and amlodipine (BDL-89 µg/kg) were detected in the
medium concentration range (BDL-874 µg/kg). The possible reason
might be the more production of diclofenac on the day of sampling.
Sparfloxacin (BDL-60 µg/kg), rosuvastatin (BDL-46 µg/kg) and gemi-
floxacin (BDL-1.6) were detected in the lowest concentration range of
BDL-60 µg/kg. Fig. 2a showed that amlodipine, diclofenac, ibuprofen,
naproxen and rosuvastatin were the most abundant PCs in the solid
waste samples with high detection frequency (80–100%). Ciprofloxacin
and ofloxacin were detected at few sampling sites with an average
detection frequency (60%) whereas, gemifloxacin, moxifloxacin, para-
cetamol and sparfloxacin were found with below average detection
frequency (20–40%). These results clearly showed that PCs of Group 1
(average detection frequency ≥75%) were present dominantly in the
solid waste samples compared with PCs of Group 2 (average detection
frequency=40%).
36
Ecotoxicology and Environmental Safety 136 (2017) 31–39
3.4. Occurrence of PCs in soil
Ten target PCs were detected in the soil samples collected from
various points except gemifloxacin as shown in Fig. 2a. The concentra-
tion of these PCs ranged from BDL-610 µg/kg as presented in Fig. 5.
Ibuprofen occurred at the highest concentration in the soil samples
(321–610 µg/kg) followed by diclofenac (101–257 µg/kg) and naprox-
en (30–199 µg/kg). Paracetamol (BDL-81 µg/kg), moxifloxacin (BDL-
37 µg/kg), ofloxacin (15–30 µg/kg), ciprofloxacin (BDL-28 µg/kg),
rosuvastatin (16–24 µg/kg), amlodipine (14–22 µg/kg) and sparflox-
acin (BDL-20 µg/kg) were detected in the below average concentration
range (BDL-81 µg/kg). Fig. 2a showed that amlodipine, diclofenac,
ibuprofen, naproxen, ofloxacin and rosuvastatin were the most abun-
dant PCs in the soil samples with the maximum detection frequency
(100%). Ciprofloxacin, moxifloxacin, paracetamol and sparfloxacin
were detected with an average detection frequency range (33.30–
66.60%) whereas, whereas, gemifloxacin could not be detected at all.
These results clearly showed that PCs of Group 1 (average detection
frequency ≥90%) were present dominantly in the sludge samples
compared with PCs of Group 2 (average detection frequency=40%).
Hussain et al. (2016a) investigated the fate of antibiotics in the
receiving environment of pharmaceutical wastewater, and reported the
maximum concentrations of ciprofloxacin (3.51 µg/kg) and ofloxacin
(4.67 µg/kg) in the soil. Calderón-Preciado et al. (2011) investigated
the occurrence of PCs in soil and their potential uptake by crops in
Spain. They reported that ibuprofen (0.213 µg/kg), diclofenac
(0.201 µg/kg) and naproxen (0.262 µg/kg) were detected in soil
irrigated with pharmaceutical containing wastewater. Vazquez-Roig
et al. (2012) investigated the distribution of PCs in soil in Spain, and
reported the maximum concentrations of ciprofloxacin (0.0046 µg/kg)
and ofloxacin (0.0033 µg/kg) whereas the concentrations of diclofenac
and ibuprofen were below the detection limit. This literature review
establishes that the concentration of target PCs reported in other
countries is lower than our findings.
3.5. Ecological risk assessment
Ecological risk assessment has become critical to approximate the
ecological risk associated with PCs in the various environmental
matrices. This risk assessment approximates the harmful dose of a
PC to particular specie present in the receiving aquatic ecosystem. This
risk assessment is generally expressed in terms of risk quotient (RQ) or
hazard quotient (HQ) that is the ratio between the Predicted
Environmental Concentration (PEC) or Measured Environmental
Concentration (MEC) to the Predicted No-Effect Concentration
(PNEC) of the PCs (Bouissou-Schurtz et al., 2014). The risk ranking
criteria has been adopted from the literature (de Souza et al., 2009;
Zhao et al., 2010). According to this criterion, RQ < 0.1 implies the
minimum risk to the organisms, 0.1≤RQ≤1 implies medium risk and
RQ=1 implies high risk (Verlicchi et al., 2012a, 2012b). Table 1
presents ecological risk assessment for only seven PCs in wastewater.
The rest of four PCs were not assessed because their PNEC values were
not available. Risk assessment was carried out against different waste-
water/fresh water species using the maximum measured concentration
of particular PCs in the wastewater. In case of paracetamol, RQ values
were found to be 64, 9.2, 5.0 and 0.11 against daphnia, S. probosci-
deus, D. magna and green algae, respectively. These results warrant
high risk of paracetamol against daphnia, S. proboscideus, and D.
magna, whereas medium risk is found against green algae. RQ for
naproxen were found to be 177 and 70 against fish and Ceriodaphnia
dubia (C. dubia). These RQ values (≫1) imply quite high risk to these
species. In case of diclofenac, RQ values were found to be 26, 76 and
12,600 against fish, green algae and Oncorhynchus mykiss (O. mykiss).
All of these values are high (≫1) and can cause high risk especially
against O. mykiss. The value of ibuprofen was assessed against fish,
green algae and O. latipes and the values were found to be 1,013, 253
M. Ashfaq et al. Ecotoxicology and Environmental Safety 136 (2017) 31–39

Table 3 which were found to be 4050, 81,000, 153, 56, 4050 and 7364,
Risk assessment of different pharmaceuticals in wastewater. respectively. All these values are very high and can cause high risk
especially against P. putida. RQ values for ciprofloxacin were calculated
Compound PNEC Specie MEC Risk Referencea
(µg/L) (µg/L) quotient against fish, daphnia, green algae and Microcystis aeruginosa (M.
aeruginosa) and the values were found to be below 0.1 against fish and
Paracetamol 1 Daphnia 64 64 (Verlicchi et al., daphnia that means no risk to these species. However, the high values
2012a, 2012b)
against green algae (220) and M. aeruginosa (440) mean that cipro-
6.92 S. proboscideus 9.2 (Orias and
Perrodin, 2013) floxacin can potentially pose risk to these species. RQ values for
12.9 D. magna 5.0 (Bouissou- moxifloxacin were less than 1 against fish, daphnia and green algae
Schurtz et al. and implying that moxifloxacin could not affect these species in the
2014) receiving environment. All of the PCs except moxifloxacin pose high
583 green algae 0.11 (Escher et al.
risk (RQ > 1) against majority of the investigated species. Thus, we
2011)
expect a high level of damage to the aquatic ecology due to the release
Naproxen 2.62 Fish 464 177 (Verlicchi et al., and mixing of untreated pharmaceutical industrial wastewater with
2012a, 2012b) surface and river water (Table 3).
6.6 C. dubia 70 (Orias and
Perrodin, 2013)
4. Conclusion
Diclofenac 9.7 Fish 252 26 (Verlicchi et al.,
2012a, 2012b) In this study wastewater, sludge, solid waste and soil samples were
3.3 green algae 76 (Escher et al. analyzed for the possible occurrence of eleven human pharmaceuticals
2011)
near pharmaceutical industrial area in Lahore Pakistan. Additionally,
0.02 O. mykiss 12600 (Orias and
Perrodin, 2013) risk assessment was also carried out against different types of species
0.05 O. mykiss 5040 (Bouissou- found in wastewater/fresh water. High concentrations of these PCs
Schurtz et al. were detected in all the environmental matrices i.e. wastewater (BDL-
2014)
1673), sludge (BDL-7273 µg/kg), soild waste (BDL-6632 µg/kg), and
Ibuprofen 1.65 Fish 1673 1013 (Verlicchi et al.,
soil (BDL-610 µg/kg). Nonsteroidal anti-inflammatory drug (NSAIDS)
2012a, 2012b) such as ibuprofen (1673 µg/L, 6046 µg/kg, 1229 µg/kg and 610 µg/
6.6 green algae 253 (Escher et al. kg), diclofenac (836 µg/L, 4968 µg/kg, 6632 µg/kg and 257 µg/kg) and
2011) naproxen (464 µg/L, 7273 µg/kg, 4819 µg/kg and 199 µg/kg) were
0.2 O. latipes 8365 (Orias and
found at very high concentrations in wastewater, sludge, solid waste
Perrodin, 2013)
0.01 O. latipes 167300 (Bouissou- and soil samples, respectively. The RQ values were very high for most
Schurtz et al. of the PCs against majority of species especially for diclofenac against
2014) O. mykiss (12,600), ibuprofen against O. latipes (167,300), ofloxacin
against P. putida (81,000) and P. subcapitata (7364). These very high
Ofloxacin 0.02 V. fisheri 81 4050 (Orias and
Perrodin, 2013)
values indicate damaging risk to the listed species. The study therefore
0.001 P. putida 81000 (Agerstrand stresses the treatment of pharmaceutical industrial waste before being
and Ruden, discharged into municipal waste and river. Additionally, it is important
2010) to investigate the occurrence of PCs in crops which are irrigated by
0.53 Fish 153 (Iatrou et al.
industrial wastewater. Moreover, it would be equally important to
2014)
1.44 Daphnia 56 (Iatrou et al. check the drug resistance among various pathogens against these PCs.
2014)
0.02 Green algae 4050 (Iatrou et al. Conflict of interest
2014)
0.011 P. subcapitata 7364 (Jiang et al.
2014)
The authors have declared no conflict of interest.

Ciprofloxacin 7285 Fish 2.2 < 0.1 (Iatrou et al. Acknowledgments

2014)
3415 Daphnia < 0.1 (Iatrou et al.
Muhammad Ashfaq is thankful to Chinese Academy of Sciences for
2014)
0.01 Green algae 220 (Iatrou et al. awarding Chinese Academy of Sciences (CAS) President's International
2014) fellowship (award number 2015PE004) for Postdoctoral Researchers to
0.005 M. aeruginosa 440 (Jiang et al. carryout research at Institute of Urban Environment Xiamen, China.
2014) Muhammad Saif Ur Rehman acknowledges the financial support of
Moxifloxacin 2380 Fish 17 < 0.1 (Iatrou et al.
Higher Education Commission of Pakistan under NRPU Project No.
2014) 4547.
1210 Daphnia < 0.1 (Iatrou et al.
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