w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 9 8 e1 0 8

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Impact of in-sewer transformation on 43

pharmaceuticals in a pressurized sewer under
anaerobic conditions

a,b,
Aleksandra Jelic a, Sara Rodriguez-Mozaz a,*, Damia Barcelo
a
Oriol Gutierrez
a
Catalan Institute for Water Research (ICRA), Scientific and Technologic Park of the University of Girona, Emili
Grahit 101, 17003 Girona, Spain
b
Institute of Environmental Assessment and Water Research (IDAEA-CSIC), Jordi Girona 18-26, 08034 Barcelona,
Spain

article info abstract

Article history: The occurrence of 43 pharmaceuticals and 2 metabolites of ibuprofen was evaluated at the
Received 5 March 2014 inlet and the outlet of a pressure sewer pipe in order to asses if in-sewer processes affect
Received in revised form the pharmaceutical concentrations during their pass through the pipe. The target com-
31 July 2014 pounds were detected at concentrations ranging from low ng/L to a few mg/L, which are in
Accepted 20 September 2014 the range commonly found in municipal wastewater of the studied area. The changes in
Available online 7 October 2014 concentrations between two sampling points were negligible for most compounds, i.e.
from
10 to 10%. A higher decrease in concentrations (25e60 %) during the pass through
Keywords: the pipe was observed for diltiazem, citalopram, clarithromycin, bezafibrate and amlodi-
Pharmaceuticals pine. Negative removal was calculated for sulfamethoxazole (
66 ± 15%) and irbesartan
Wastewater (
58 ± 25%), which may be due to the conversion of conjugates back to their parent
Sewage system compounds in the sewer. The results show that microbial transformation of pharmaceu-
ticals begins in sewer, albeit to different extents for different compounds. Therefore, the
in-sewer transformation of pharmaceuticals should be assessed especially when their
concentrations are used to estimate and refine the estimation of their per capita con-
sumption in a catchment of interest in the sewage epidemiology approach.
© 2014 Elsevier Ltd. All rights reserved.

development of advanced analytical techniques and in-

1. Introduction
Pharmaceutical compounds (PhACs) have been given much
attention in the last decade due to their documented presence
in the environment and concern about their potential adverse
effects to humans and wildlife. This was largely owing to the
struments, which enabled the trace-level detection of these
compounds in different environmental compartments
including surface and groundwater, sediment and soil
(Kümmerer, 2009). The issue of “pharmaceuticals in the
environment” has only been covered by regulations developed
by the Swiss environmental agency for Switzerland (Eggen

* Corresponding author. Tel.: þ34 972 18 33 80.

E-mail address: srodriquez@icra.cat (S. Rodriguez-Mozaz).
http://dx.doi.org/10.1016/j.watres.2014.09.033
0043-1354/© 2014 Elsevier Ltd. All rights reserved.
 w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 9 8 e1 0 8
et al., 2014), but it has been recognized as an emerging prob-
lem in the Directive 2010/84/EU and Regulation 1235/2010 on
pharmacovigilance that recommend the monitoring and
evaluation of the risk of environmental effects of medicinal
products.
Within the Urban wastewater system (UWS), wastewater
treatment plants (WWTP) have been identified as a point of
discharge of pharmaceuticals into the environment.
Numerous studies have been conducted to assess the
magnitude of pharmaceuticals that reach the environment
through the wastewater and sludge discharge, and to evaluate
the efficiency of typical wastewater treatment plants
regarding the removal of these compounds from raw waste-
water (Jelic et al., 2011; Joss et al., 2005; Radjenovic et al., 2009).
The wastewater quality and its pharmaceuticals content,
particularly, have been typically monitored at the influent
and/or the effluent of a treatment plant, where these micro-
pollutants were detected at concentrations ranging from low
ng/L to a few mg/L (Verlicchi et al., 2012). A wide variation in
removal efficiencies has been reported for individual com-
pounds in separate studies as well as across therapeutic
classes and treatment processes (Onesios et al., 2009).
The actual and major source of PhACs is households, up-
stream of the catchment, where pharmaceuticals are
consumed and eventually released to the sewer systems.
Sewers are underground infrastructure composed by drains,
pipes and pumping stations that transport wastewater to the
treatment facilities (Hvitved-Jacobsen, 2002). Two types of
sewer pipes are found depending on the topography: 1) gravity
pipes, where wastewater flows naturally from higher to lower
elevation in partially-filled sections, and 2) pressure pipes
(force mains) e completely-filled tubes e with a pump station
at their inlet to mechanically push sewage through flat and
sloping terrains. Sewers have been traditionally considered as
just a sewage collection and transport system, but in fact they
are also active chemical and biological reactors (Huisman
et al., 2002; Rudelle et al., 2011; Schilperoort et al., 2012). In
gravity sewers, having a relatively long transport time, a sig-
nificant reduction in the amount of biodegradable substrate
and production of biomass may occur under aerobic condi-
tions (Hvitved-Jacobsen et al., 2002). Completely-filled pres-
sured pipes are prone to development of septic conditions
which make them preferred grow-locations for anaerobic
biofilms that generate detrimental compounds such as sulfide
(H2S), volatile organic sulfur compounds (VSOC) and methane
(CH4) (Guisasola et al., 2008; Gutierrez et al., 2010; Hvitved-
Jacobsen et al., 2013; Sutherland-Stacey et al., 2008).
Micropollutants in sewers are still scarcely reported in the
literature. Microbial and chemical processes that occur in
sewers have been generally neglected. This issue has been
recognized in the field of sewage epidemiology, which basi-
cally aims at estimating the actual illicit drug use and trends
from the concentrations detected in influent wastewater
(Daughton, 2001). It has been emphasized that the main limi-
tation of the approach, which directly influences its accuracy,
is the lack of comprehensive data on drug marker stability in
sewer systems and during storage (Castiglioni et al., 2012; Khan
and Nicell, 2011; van Nuijs et al., 2011). The stability of illicit
drugs during sample storage has been assessed in several
studies (Baker and Kasprzyk-Hordern, 2011; Castiglioni et al.,
99
2011; van Nuijs et al., 2012). Lai et al. (2011) quantified the
overall uncertainty of the sewage epidemiology approach and
included five prescription pharmaceuticals (atenolol, carba-
mazepine, gabapentin, hydrochlorothiazide and venlafaxine)
to refine the estimation of the number of people that contrib-
uted to wastewater in the monitored catchment. However,
possible in-sewer transformation of the pharmaceuticals was
not considered. Only Thai et al. (2014) have studied how in-
sewer transformation processes alter the concentrations of
organic pollutants, in particular illicit drugs and their metab-
olites, under different conditions, at laboratory-scale sewer
reactors. They found that the biofilms in the lab-scale sewer
have significantly enhanced the degradation rate of some
compounds (i.e. cocaine, and a metabolite of heroin, 6-acetyl
morphine) in comparison to the rates in wastewater only,
and they suggested that the fate of illicit drugs will depend on
sewer conditions. As mentioned before, pressure pipes are the
most biologically-active sections of sewer networks due to
higher biofilm presence and biofilm-sewage contact (Hvitved-
Jacobsen, T., 2002). Under anaerobic conditions in
completely-filled pipes, septic biofilm grows attached to the
sewer walls. Sewer biofilm captures the compounds dissolved
in wastewater and change their concentrations in the flowing
sewage water (Huisman et al., 2002). In addition, for a similar
length of pipe, hydraulic retention time (HRT) in pressure pipes
is typically longer than in gravity sections since pressure pipes
need to be completely filled and act as a plug flow reactor
needing higher volumes to reach similar distances. This can
lead to higher biofilm-sewage contact time that can promote
the transformation processes. To date, there has been no
comprehensive study on whether in-sewer biotransformation
of pharmaceuticals occurs and to which extent.
In this work, we aimed at assessing the occurrence and the
extent of biotransformation of 43 pharmaceuticals and 2 me-
tabolites of ibuprofen in pressure sewer pipes. The study was
carried out at a flat 7620 m long pipe transporting domestic
wastewater from the municipality of Sant Pere Pescador (Cat-
alonia, Spain). The target compounds belong to commonly
prescribed and over-the-counter pharmaceuticals (i.e. nonste-
roidal anti-inflammatory agents and analgesics, lipid modi-
fying agents, psycholeptic and antiepileptic drugs etc.) and
were selected on the basis of their high consumption and/or
frequently reported detection in wastewaters, and the possi-
bility to be analyzed under the same experimental conditions.
2. Material and methods
2.1. Chemicals
All pharmaceuticals and their corresponding isotope-labeled
standards were of high purity grade (>90%). Detailed infor-
mation on the chemicals used, the providers of the analytical
standards, and the preparation of the mixture solutions is
given in the Supporting Information (Table S1).
2.2. Description of the sewer system
The studied collection system is situated in Sant Pere Pesca-
dor, a small catchment in the north-east coastal region of
100 w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 9 8 e1 0 8
Catalonia (Spain) (Fig. 1). The system consists of a pump sta-
tion (Point 1) that collects the wastewater of 3000 inhabitants
during winter, and up to around 35,000 during summer
months. Estimated population in the sampling period was
7500 inhabitants. The pump station at Point 1 collects both
raw sewage from a close neighborhood (HRT 0.5 h) and from 2
pressure pipes upstream of the catchment with average HRTs
of 1.5 and 2 h. The average volume of sewage pumped per
event is 23.3 m3, where pump events last around 4 min. The
wastewater is pushed through the pressure pipe until l’Escala
WWTP (Point 2). The length of the sewer pipe is 7620 m, and its
internal diameter is 500 mm. The average wastewater flow
was 62.7 m3/h, which resulted in an HRT of 21 ± 2 h (more
details in Supplementary Information). The system has no
lateral streams and is in good condition, therefore infiltration
and exfiltration can be considered negligible. Wastewater
characteristics were typical of domestic sewage. Dissolved
concentrations of sulfate (SO2
4 ) and sulfide (H2S) were used as
indicators of the biological activity of the anaerobic sewer
biofilm (Supporting information S3).
2.3. Sampling
Raw sewage water samples were collected daily as twenty-
four hour composite samples at the pumping station e Point
1 (Fig. 1) and at the influent of WWTP L'Escala (Point 2, Fig. 1).
The start of sampling at Point 2 (i.e. WWTP influent) was al-
ways delayed for 21-hour (i.e. HRT of the pipe) in order to
capture the portion of the wastewater stream measured at
Point 1. The sampling was carried out over five consecutive
days in July 2013. The weather was dry and stable, with the
environmental temperatures ranging from 14 to 30  C. Time
proportional samples were collected employing 2 portable
automatic refrigerated samplers Hach-Lange Bühler BL 2000
with 24 PE containers of 1 L. The autosamplers were pro-
grammed to collect a 50 mL sample each 10 min to form a
7.2 ± 0.4 L composite daily. The frequency of pump events and
the HRT variation are shown in Figure S1 (Supp. Information).
Fig. 1 e Location and layout of the studied sewer. Point 1: pump station collecting wastewater from the Sant Pere Pescador
municipality; Point 2: wastewater treatment plant.
The sampler tube at Point 1 was installed deep in the pump-
station wet well to ensure the collection wastewater sam-
ples, it contained always at least 5.5 m3 of raw wastewater.
The samples at Point 2 (outlet) were taken from a tap installed
5 m before the discharge to WWTP and that always contained
around 1 m3 wastewater. Collected wastewater, i.e. 4 inlet and
4 outlet samples, were maintained at 4  C to avoid biological
degradation. The samples were extracted and processed
within 12 h, and analyzed immediately after the preparation.
2.4. Sample preparation and analysis
Samples for the analysis of PHACs, i.e. 4 inlet samples and 4
outlet wastewater samples, were filtered through a 1-mm glass
fiber filter and a 0.45-mm nylon fiber filter (Whatman, U.K.). A
Na2EDTA solution (0.1 M) was added to 50 mL aliquots of the
filtered wastewater samples (all in triplicate) to a concentra-
tion of 0.1% (g solute/g solution). Then, the samples were
spiked with a solution of surrogate standards to a concen-
tration of 200 ng/L and preconcentrated by solid phase
extraction e Oasis® HLB (Waters, Milford, MA, USA), using a
Baker vacuum system (J.T. Baker, Deventer, The Netherlands).
Analytes were eluted by 6 mL of pure methanol and the ex-
tracts were gently dried under nitrogen and reconstituted in
1 mL of a methanol/water mixture (10/90, v/v) and fortified by
a mixture of internal standards (Table S1) to a final concen-
tration of 20 ng/mL. The samples were analyzed by a Waters
Acquity Ultra-PerformanceTM liquid chromatography system
(Waters, Milford, MA, USA) coupled to a 5500 QTRAP hybrid
triple quadrupole-linear ion trap mass spectrometer (Applied
Biosystems, Foster City, CA, USA) with a turbo Ion Spray
source, according to a previously reported method (Gros et al.,
2012).
Quantitative analysis was performed by internal standard
approach, and two isotope-labeled compounds, i.e. sulfadox-
ine-d3 for positive ionization mode, and ketoprofen-d3 for
negative ionization mode, were added to all the samples
before the extraction as control standards. Linear least
Table 1 e Method performance characteristics, i.e. method quantification limits (MQL), matrix effect (given as a percentage of signal suppression), average recovery,
intraday precision, standard error (SE) of replicate measurements, uncertainty associated with chemical analysis, and the day-to-day variation in concentrations of the
target pharmaceuticals.
Therapeutic group Compound MQL Matrix effect Recovery Intraday SE of replicate Uncertainty Day-to-day
(mean, n ¼ 6) precision measurements associated variation in
with chemical concentrations
analysis
ng/L % % % % % %
(Mean, n ¼ 6) (Mean, (Mean, (SE, n ¼ 6) (n ¼ 9) Inlet Outlet
n ¼ 6) n ¼ 6)
Nonsteroidal anti-inflamatory Ketoprofen 4 12 111 6 5 8 2 5
agents/Analgesics (M01) Naproxen 27 50 66 6 3 7 10 4
Ibuprofen 37 15 105 6 5 8 8 10
Indomethacin 12 32 52 6 10 11 12 19
Diclofenac 10 32 67 4 5 7 8 11
Propyphenazone 4 60 69 3 7 8 58 59

w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 9 8 e1 0 8
Oxycodone 5 38 104 4 9 10 12 8
Codeine 14 28 117 4 6 7 14 11
Lipid regulators (C10) Bezafibrate 20 38 109 1 8 8 41 32
Gemfibrozil 15 40 110 6 3 7 15 6
Pravastatin 9 22 89 4 3 6 7 8
Fluvastitatin 9 13 40 3 2 4 5 4
Atorvastatin 7 17 39 3 4 6 8 13
Antiepileptics (N03) Carbamazepine 14 80 108 1 2 3 13 22
Psycholeptics (N05) Lorazepam 10 28 107 3 7 8 9 13
Psychoanaleptics (N06) Citalopram 10 70 115 5 7 9 5 9
Venlafaxine 14 50 122 2 4 5 13 8
Drugs for peptic ulcer and Ranitidine 12 32 106 6 10 12 15 8
gastro-oesophageal reflux Cimetidine 10 50 123 3 6 7 14 29
disease (A02)
Beta-blocking agents (C07) Atenolol 5 13 91 3 4 5 9 11
Sotalol 7 70 77 1 6 6 13 6
Propanolol 11 81 82 4 10 11 21 8
Metoprolol 7 70 90 3 5 6 7 4
Diuretic (C03) Furosemide 14 62 85 3 6 7 7 12
Torasemide 4 60 122 8 5 10 12 16
Antidiabetic (A10) Glibenclamide 12 78 64 5 4 7 1 3
Agents acting on the Amlodipine 10 81 78 4 9 10 15 9
renin-angiotensin system Losartan 19 46 45 4 5 7 14 13
(C09) Irbesartan 5 10 58 5 10 11 12 4
Valsartan 5 30 38 7 2 7 17 16
Antithrombotic agents (B01) Clopidogrel 4 78 62 3 7 8 12 7
Drugs for obstructive airway Salbutamol 4 63 74 3 4 6 21 22
diseases (R03)
(continued on next page)

101
102 w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 9 8 e1 0 8

squares regression calibration curves were constructed using

Outlet
concentrations
variation in 9 calibration standard mixtures of the target compounds at
Day-to-day

16
12
10
8
13
8
17
22
10
4
29
7
4
concentrations ranging from 0.05 to 200 ng/mL, and for all the

%
compounds it was linear over the range from 0.1 to 100 ng/mL.
Method quantification limits (MQLs) were calculated as ten
Inlet

34
8
13
9
17
15
23
27
7
12
31
6
4
times signal-to-noise, and the values are reported in Table 1.
To determine the relative recoveries of the analyzed com-
pounds during the applied method, six wastewater samples
with chemical
Uncertainty
associated

were spiked with the pharmaceutical standard mixture to a

analysis

final concentration of 50 ng/mL. Since the samples may

11
8
10
9
9
11
4
5
8
7
10
6
7
%

contain target compounds, blanks (non-spiked samples) were

also analyzed and their quantified concentrations were sub-
tracted from those of spiked samples. The recoveries and the
standard errors (n ¼ 6, confidence interval (CI) 68%) are sum-
measurements
SE of replicate

marized in Table 1. Instrumental intraday precision of the

applied method was calculated from 6 injections of a standard
(n ¼ 9)

8
7
9
8
7
8
3
4
7
6
8
4
2
%

solution at 50 ng/mL over the course of day. It is reported as

the standard error of the mean (n ¼ 6, 68% CI) in Table 1.
Analytical methods for the monitoring of parameters
related to anaerobic activities in sewer systems included the
analysis of dissolved sulfur species, volatile fatty acids (VFAs),
(SE, n ¼ 6)
precision
Intraday

chemical oxygen demand (COD), total and volatile suspended

%

7
4
4
5
4
7
2
2
4
4
5
4
6

solids (TSS and VSS, respectively), pH, conductivity, temper-

ature and dissolved oxygen (DO). For the analysis of dissolved
sulfur species, 1.5 mL of wastewater was filtered through a
0.22 mm filter and enriched by 0.5 mL preserving solution of
Recovery

(Mean,
n ¼ 6)

sulfide anti-oxidant buffer (SAOB) (Keller-Lehmann et al.

87
80
57
107
82
110
96
73
73
72
48
115
75
%

2006). Samples were analyzed by an ion chromatograph (IC)

with UV and conductivity detector (Dionex ICS-5000). Online
sulfide measurements were carried out by an UVeVIS spec-
(mean, n ¼ 6)
Matrix effect

trometer probe s::can spectro::lyser (Messtechnik, GmbH,

(Mean,

Austria) according to the method of Sutherland-Stacey et al.

n ¼ 6)
90
10
80
48
61
72
50
70
90
80
80
10
15
%

(2008). VFAs were measured by gas chromatography coupled

to FID detector (Thermofisher Scientific). COD analysis was
performed using a standard photometric test kit with
commercially available reagent (LCK 114, Hach Lang). Absor-
(Mean, n ¼ 6)

bance readings were conducted on DR 2800 Hach Langue

spectrometer (method 814). TSS and VSS were analyzed ac-
MQL

ng/L

21
5
16
10
7
7
8
7
30
5
10
35
37

cording to the standard method 2510D (APHA 1998). Temper-

ature, pH, conductivity, and DO concentration were measured
by Portable probes Unisense Model YSI Digital Pro.
1-hydroxy Ibuprofen
2-hydroxy ibuprofen

2.5. Uncertainty estimation

Sulfamethoxazole
Compound

Clarithromycin

Thiabendazole
Trimethoprim
Azithromycin
Erythromycin

The uncertainty associated with the measured PhACs con-

Ciprofloxacin
Tetracycline

Levamisole

centrations was calculated from the individual uncertainties

Diltiazem
Ofloxacin

from PHACs chemical analysis and from sampling.

The uncertainty of chemical analysis was estimated from
the relative recoveries (six spiked samples), triplicate analysis
of the samples, intra-day instrumental precision (six in-
Calcium channel blockers (C08)

jections of standard at 50 ng/mL every 4 h) and other uncer-

Antibacterials for systemic

tainty factors (i.e. 2%, (Kovalova et al., 2012)). The mixture of

Table 1 e (continued )

chemical standards was prepared just before the analysis, so

Ibuprofen metabolites
Therapeutic group

the error associated with the stability of the solution could be

Antihelmintic (P02)

considered negligible.
The uncertainty due to sampling was estimated by
use (J01)

considering the population served by the sewer and their

pharmaceutical consumption pattern, and the applied sam-
pling procedure as proposed in the literature (Castiglioni et al.,
2012; Ort et al., 2010a, 2010b; Weissbrodt et al., 2009). The
 w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 9 8 e1 0 8
population number and their pharmaceutical consumption
pattern figure in the expected Number of wastewater pulses
containing the compounds of interest (Np), and it was calcu-
lated according to Ort et al. (2010b). The Np may not represent
realistic pharmaceuticals consumption pattern for the
catchment area, but is useful to get a general idea. The sewer
system served approx 7500 population equivalents during the
sampling period, calculated from the actual wastewater flow
data (i.e. 1505 ± 26 m3/day) by assuming that a person pro-
duces 200 L of wastewater per day. The consumption data
were obtained from the technical reports published by the
Spanish Agency of Medicines and Healthcare Products
(Agencia Espan ~ ola de Medicamentos y Productos Sanitarios).
The consumption data and the calculated Np, together with
the assumed values of sampling uncertainty are provided as
Supp. Information (Section S4).
3. Results and discussion
3.1. Concentrations of PhACs in the pressure sewer pipe
Concentrations of the target pharmaceuticals, 43 pharma-
ceuticals and 2 metabolites of ibuprofen, i.e. 1-hydroxy-
ibuprofen (1-OH-Ibuprofen) and 2-hydroxy-ibuprofen (2-OH-
Ibuprofen), in analyzed wastewater samples ranged from low
ng/L (e.g. 6e7 ng/L of glibenclamide) to a few mg/L (e.g. 4e6 mg/L
of ibuprofen). Fig. 2 summarizes the concentrations of the
compounds at the inlet and the outlet of the studied pressure
pipe during four consecutive days. The figure shows the daily
concentrations with associated uncertainties in the analytical
measurement (error bars). Sampling uncertainty was not
included in the error bars for a clearer picture of the changes
in pharmaceuticals concentrations, and the compounds with
sampling uncertainty larger than 20% are underlined. Day-to-
day variations in the measured pharmaceuticals concentra-
tions were between 3 and 16% for 80% of the analyzed com-
pounds, at both sampling points (Table 1). Higher day-to-day
variations in concentrations (20e59%) were observed for an-
timicrobials erythromycin, sulfamethoxazole and trimetho-
prim, lipid modifying drug bezafibrate, anti-acid drug
cimetidin, and antifungal drug thiabendazole. This may be
associated with the consumption of these pharmaceuticals in
the studied area, which is connected with the results in a way
that lower consumption introduces higher sampling uncer-
tainty (Supporting information, Table S4). If these compounds
are expected to be excreted in only a few wastewater pulses
daily, then their concentrations, detected in the samples
taken by the employed sampling protocol, may easily be
underestimated (Ort et al., 2010a).
Non-steroidal anti-inflammatory drugs ibuprofen and
naproxen, and antihypertensive drug valsartan were detected
at the highest concentrations at both sampling points.
Average concentrations at the inlet and the outlet of the sewer
pipe were 5.3 and 4.9 mg/L for ibuprofen, 3.5 and 3.4 mg/L for
naproxen, and 1.9 and 1.9 mg/L for valsartan, respectively.
These concentrations are in the same range as those previ-
ously reported for WWTP influents of the studied area
(Ferrando-Climent et al., 2012; Gros et al., 2012). A human
metabolite of ibuprofen, 2-hydroxy-ibuprofen, was detected
103
at similar concentrations as its parent compound, i.e. 5.1 and
5.0 mg/L at the inlet and the outlet of the sewer, respectively.
Quantified ratio of 2-OH-ibuprofen to ibuprofen (i.e. 1:1 in
average) at the inlet was lower than a typical human excretion
ratio of 1.7:1 (Lienert el al., 2007; Weigel et al., 2004), and it was
the same at the outlet. The proportion varied from study to
study (Ferrando-Climent et al., 2012; Weigel et al., 2004), which
may be due to stereoselective metabolism of ibuprofen (Rudy
et al., 1991) and its administration routes (i.e. oral and topical,
whereas the latter introduces active, unmetabolised, com-
pound to sewer (Daughton and Ruhoy, 2009)). Day-to-day
variation in the concentrations of the given compounds
ranged from 4% (ibuprofen) to 17% (valsartan) for inlet
wastewater, and from 4% (naproxen and 2-OH-ibuprofen) to
16% (valsartan). Based on consumption data and the applied
sampling protocol, we assumed that the sampling uncertainty
for these compounds was less than 5e10 % (CI 68%) on indi-
vidual days.
The lowest concentrations (<20 ng/L) were quantified for
diuretic torasemide, antithrombotic agent clopidogrel, and
antidiabetic glibenclamide, anthelmintic levamisol, and an-
algesics oxycodone and propyphenazone. According to the
consumption data for Spain (Supp. Info, Table S3), we could
predict to find torasemide, clopidogrel and glibenclamide at
higher concentrations. A plausible explanation lies in their
extensive metabolism, but, while clopidogrel and glibencla-
mide are almost completely metabolized (Sangkuhl et al.,
2010; Sundaresan et al., 1997), torasemide is excreted by 25%
as unchanged compound (Begon ~ a Barroso et al., 2001). This
indicates that the national data on pharmaceuticals con-
sumption are not directly transferable to local levels, and
especially not to small catchments; and the sampling uncer-
tainty may exceed the levels expected from the consumption
data. Therefore, when “predicting”, we should have in mind
that concentrations of pharmaceutical residues in WWTP in-
fluents will depend not only on the route of administration
(oral/topical) and the metabolism of pharmaceuticals, but also
on the size and pharmaceuticals consumption habits of a
given settlement population, and on wastewater sources, i.e.
residential, university, hospital etc.
3.2. Aqueous removal of PhACs
Sewer pipes could be considered as an anaerobic reactor,
where various microbial, chemical and physicochemical pro-
cesses occur simultaneously, and thus some changes in
pharmaceuticals concentrations could be expected (Hvitved-
Jacobsen et al., 2013). Fig. 3 shows the increase of sulfide
concentrations in wastewater from Point 1 (inlet) to Point 2
(outlet) of the sewer pipe, which indicates high microbial ac-
tivity in the sewer. Although the transformation of the organic
matter in sewer occurs due to biological activity of microbial
communities in both the wastewater stream and biofilm,
Gutierrez et al. (2009) showed that the planktonic microor-
ganisms had a negligible contribution to the overall sulfide
bioproduction (approximately <2% of the overall rate), indi-
cating the predominance of sewer wall biofilms. Similarly,
Thai et al. (2014) showed that biofilm microbial activity
increased the hydrolysis of cocaine compared to the sus-
pended microbes in the wastewater alone. It can thus be
104 w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 9 8 e1 0 8

Fig. 2 e Concentrations of the target pharmaceuticals detected in the sewer pipen: the gray and white rhombi show average
daily concentrations (n ¼ 3) at the inlet and the outlet of the pipe, respectively, and the error bars represent the analytical
measurement uncertainty associated with the reported concentrations (CI 68%). Underlined are the compounds with an
assumed sampling uncertainty of >20% (no information on the consumption of the compounds underlined with dashed
line).

anticipated that biofilm bacteria may affect the in-sewer
concentrations of pharmaceuticals as well, as they spend
around 21 h in the sewer.
Fig. 4 depicts the average aqueous removal (%, circle
markers) of the target PhACs during their pass through the
sewer pipe. The error bars denote the minimum and
maximum removal efficiencies for the sampling days. The
term “removal” here refers to the pharmaceuticals reduction
from the inlet to the outlet of the pipe, due to a combination of
different processes: chemical and physical transformation,
biodegradation and sorption to solid matter. The removal was
calculated from concentration values since the day-to-day
 w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 9 8 e1 0 8 105

70.0

60.0

50.0
H S concentration (mg/L)

H2S Point 1
H2S Point 2
40.0 H2S Point 1 online
H2S Point 2 online
30.0

20.0

10.0

0.0
00:00 01:00 02:00 03:00 04:00 05:00 06:00 07:00 08:00 09:00 10:00 11:00 12:00 13:00 14:00 15:00 16:00 17:00 18:00 19:00 20:00 21:00 22:00 23:00 00:00
Time (h)

Fig. 3 e Sulfide concentrations at Point 1 and Point 2 of the system. Increase of H2S indicates strong anaerobic biological
activity.

variation in the average daily wastewater flow was small
during the sampling period. Our results are based on the
analysis of the aqueous fraction of wastewater samples
because considerable sorption onto suspended solids could be
expected only for the antibiotics azithromycin, ciprofloxacin
and tetracycline, according to the literature values of sorption
coefficients of the target compounds for primary/secondary
sludge (Supporting information, Table S5).
For most compounds, the average removal, calculated
pair-wise (influent-effluent, for each sampling day) and then
averaged, ranged from
10 to 10%, which could be considered
negligible when compared with the overall uncertainty asso-
ciated with the concentration values. Slightly higher removal
was observed for calcium channel blocker diltiazem (23%;
12e31% (mean; minemax)), antidepressant drug citalopram
(25%; 11e36 %), antibiotic clarithromycin (26%; 22e31%) and
ciprofloxacin (21%; 2e31%) and lipid modifying drug bezafi-
brate (32%; 12e62%). However, because of relatively high
sampling uncertainty and/or day-to-day variation in removal
efficiencies of the compounds, these percentages should be
taken as rough estimates of removal. The highest average
aqueous removal was estimated for antihypertensive
amlodipine, i.e. 59% (54e67 % (minemax)). The removal of
citalopram, bezafibrate, diltiazem, clarithromycin and amlo-
dipine could be attributed mainly to biochemical processes,
except for ciprofloxacin, for which sorption to organic par-
ticulate matter cannot be neglected.
A significant increase in the concentrations (“negative
removal”) of sulfamethoxazole (
66 ± 15%) and irbesartan
(
58 ± 25%) were observed during the pass through the sewer
pipe. The increase in the concentrations may be due to the
cleavage of the conjugates and/or re-transformation of me-
tabolites by microbial community present in sewer waste-
water. The known metabolism of sulfamethoxazole in
mammals involves glucuronidation to N1-glucuronide conju-
gate (9e15 %) and acetylation to N4-acetyl-sulfamethoxazole
(43%) (van der Ven et al., 1995). The re-transformation of these
two metabolites to sulfamethoxazole was observed during
wastewater treatment (Gobel et al., 2005) and in water-
sediment tests (Radke et al., 2009). Additionally, Radke et al.
(2009) reported that the elimination of the sulfamethoxazole
and its metabolites in the water sediment tests was due to
cometabolic microbial degradation, whereas abiotic processes
like hydrolysis or sorption to the sediment were of minor

Fig. 4 e Removal of pharmaceuticals during the pass through the pressure sewer pipe (B: average value, calculated pair-
wise (influent-effluent, for each sampling day) and then averaged; in gray are the compounds with the sampling
uncertainty higher than 20%; error bars: minimum and maximum removal efficiencies for each sampling day).
106 w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 9 8 e1 0 8
importance for their fate. Irbesartan is excreted by 5% as un-
changed drug in urine, with the main metabolites being
omega-1-hydroxylated metabolite (25%) and its glucuronide
conjugate, tetrazole N2-glucuronide (10%) (Chando et al.,
1998). Therefore, we could assume that the “negative”
removal was due to the cleavage of the conjugate by enzymes
produced by wastewater bacteria (e.g. b-glucuridase from
Escherichia coli (Bitton, 1994)), as in the case of sulfamethoxa-
zole. Average negative removal was also calculated for anti-
biotic erythromycin (
30%;
97 to 9%), antifungal agent
thiabendazole (
2%;
82 to 55%), and NSAID indometacin
(
20%;
75 to 23%), with very high day-to-day variations
observed in the removals. This may indicate that even though
the employed sampling frequency was quite high (every
10 min) it might not be high enough to catch very scarce
wastewater pulses that contained these pharmaceuticals, due
to their low consumption (Supp. Information, Table S3).
The target compounds exhibit a wide range of physico-
chemical properties (Supp. Info, Table S2), and these affect their
biodegradability and sorption, and thus govern their fate during
the pass through the sewer pipe. Literature studies show a wide
variation in the removal of pharmaceuticals during different
wastewater and sludge treatment processes (Mie ge et al., 2009;
Onesios et al., 2009). It is difficult to draw a clear conclusion on
the fate of pharmaceuticals that belong to the same medicinal
groups, as they are classified mainly according to their thera-
peutic application. In this study, we observed similar removal
(in average negative) for beta-blockers atenolol, sotalol and
metoprolol, which are characterized by comparable sorption
and biodegradation characteristics under aerobic conditions,
i.e. low sorption (<0.04 L/gCOD) and partial degradation (25e90%)
(Maurer et al., 2007). Propranolol is the only beta-blocker here
that shows affinity to primary sludge (Radjenovic et al., 2009),
that could be a reason for its low, but positive removal of 17%
(5e35%). Also in the group of angiotensin II receptor antago-
nists, we observed comparable, negative removal for irbesartan
and losartan that have similar physicochemical properties
(Table S2) and form N2-tetrazole glucuronides (Chando et al.,
1998; Iwamura et al., 2011).
3.3. Future prospects
This is, to our knowledge, the first study that investigated the
extent of biotransformation of pharmaceuticals under septic
conditions in real pressure sewer. The importance of consid-
ering real sewer conditions is underlined in a study of Thai et al.
(2014) on degradation of illicit drugs in lab-scale sewer reactors
under pressure (rising) pipe and gravity sewer conditions. They
concluded that the factors such as A/V ratio (i.e. ratio of area of
biofilms/volume of wastewater in the sewer), flowing condi-
tions, HRT etc, should be considered when extending the re-
sults from a lab-scale study to real sewer systems. The results of
our study showed that most target pharmaceuticals arrived
unchanged, or slightly changed, to the WWTP influent after 21 h
of passage through the pressure pipe. These compounds may
be used as stable markers in the sewage epidemiology
approach, for the refining of the number of population served
by a WWTP when estimating drug use and trends (O'Brien et al.,
2014). Further study about pharmaceuticals in gravity sections
is needed, thus to cover two major scenarios in sewer systems e
both gravity and pressure pipes -and obtain more accurate drug
consumption estimates.
Another issue to consider is the biotransformation pathway
of pharmaceuticals during their pass through sewer system.
The few studies that have examined the fate of pharmaceuti-
cals under anaerobic conditions, in anaerobic digestion of
sewage sludge and wastewater at laboratory-scale (Carballa
et al., 2007; de Graaff et al., 2011), showed that pharmaceuti-
cals are either not removed or they are biotransformed at a
slower rate than during aerobic treatments (de Graaff et al.,
2011; Ericson, 2007). Similar conclusions were drawn in our
study, although the comparison is not straightforward as the
characteristics of wastewater and activity of its microbial
community under septic conditions in the pressure pipe, its
configuration and the SRT (SRT ¼ HRT ¼ 21 h) differ much from
the commonly studied treatments. Still, the transformation
mechanisms of pharmaceuticals under anaerobic conditions
and the occurrence and fate of the transformation products
need better understanding. Which removal mechanism, i.e.
physical, chemical and/or biological contributes mostly to the
transformation of a parent compound, and to which degree it
can be transformed can only be evaluated under lab-scale
controlled studies. For our work, a future study should
consider the biotransformation mechanism of the compounds
such as sulfamethoxazole and irbesartan, to confirm if the
“negative removal” can be undoubtedly attributed to the back-
transformation of their conjugates.
4. Conclusions
The present study evaluated the effect of the in-sewer pro-
cesses on pharmaceuticals during their pass through a pres-
sure sewer pipe. These are the major findings:
- The change in the concentrations was negligible for most
of the analyzed compounds (i.e.
10 to 10%) under the
anaerobic conditions in the sewer pipe.
- An increase in the concentrations of sulfamethoxazole
(
66 ± 15%) and irbesartan (
58 ± 25%) was observed dur-
ing the pass through the sewer. This may be due to the
enzymatic and/or chemical cleavage of conjugates of these
compounds in raw wastewater that liberates the parent
compounds, and thus, for more accurate estimation of
overall biotransformation, the concentrations of the con-
jugates should be considered along with those of the
parent compounds.
- The assessment of in-sewer biotransformation of phar-
maceuticals and the choice of adequate drug marker (i.e.
stable and non-sorptive) is very important when applying
sewage epidemiology approach to estimate drug con-
sumption trends.
Acknowledgments
The study is a part of the project CTM 2011-27163 financed by
the Spanish Ministery of Economy and Competitiveness. It
 w a t e r r e s e a r c h 6 8 ( 2 0 1 5 ) 9 8 e1 0 8
has been co-financed by European Union through the Euro-
pean Regional Development Fund (ERDF) and partly supported
by the Generalitat de Catalunya (Consolidated Research
Group: Catalan Institute for Water Research 2014 SGR 291). OG
acknowledges the support of the European Commission Marie
Curie program (Project 2010-RG277050). DB acknowledges
support from the Visiting Professor Program of the King Saud
University. The authors thank to Consorci Costa Brava e
Empresa Mixta Aigües Costa Brava for their help and collab-
oration in the samplings.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.watres.2014.09.033.
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