water research 44 (2010) 3214–3224

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Removal of Pharmaceutical and Personal Care Products

(PPCPs) under nitrifying and denitrifying conditions

Sonia Suarez, Juan M. Lema, Francisco Omil*

School of Engineering, University of Santiago de Compostela, 15782 Santiago de Compostela, Galicia, Spain

article info abstract

Article history:
Received 21 September 2009
Received in revised form
17 February 2010
Accepted 27 February 2010
Available online 6 March 2010
Keywords:
The contribution of volatilization, sorption and transformation to the removal of 16
Pharmaceutical and Personal Care Products (PPCPs) in two lab-scale conventional activated
sludge reactors, working under nitrifying (aerobic) and denitrifying (anoxic) conditions for
more than 1.5 years, have been assessed. Pseudo-first order biological degradation rate
constants (kbiol) were calculated for the selected compounds in both reactors. Faster degra-
dation kinetics were measured in the nitrifying reactor compared to the denitrifying system
for the majority of PPCPs. Compounds could be classified according to their kbiol into very
highly (kbiol > 5 L g1 1 1 1 1 1
SS d ), highly (1 < kbiol < 5 L gSS d ), moderately (0.5 < kbiol < 1 L gSS d )

Fragrances and hardly (kbiol < 0.5 L g1 1

SS d ) biodegradable.

Hormones Results indicated that fluoxetine (FLX), natural estrogens (E1 þ E2) and musk fragrances
Pharmaceuticals (HHCB, AHTN and ADBI) were transformed to a large extent under aerobic (>75%) and
Redox conditions anoxic (>65%) conditions, whereas naproxen (NPX), ethinylestradiol (EE2), roxithromycin
Solid retention time (ROX) and erythromycin (ERY) were only significantly transformed in the aerobic reactor
Temperature (>80%). The anti-depressant citalopram (CTL) was moderately biotransformed under both,
aerobic and anoxic conditions (>60% and >40%, respectively). Some compounds, as car-
bamazepine (CBZ), diazepam (DZP), sulfamethoxazole (SMX) and trimethoprim (TMP),
manifested high resistance to biological transformation.
Solids Retention Time (SRTaerobic >50 d and <50 d; SRTanoxic >20 d and <20 d) had
a slightly positive effect on the removal of FLX, NPX, CTL, EE2 and natural estrogens
(increase in removal efficiencies <10%). Removal of diclofenac (DCF) in the aerobic reactor
was positively affected by the development of nitrifying biomass and increased from 0% up
to 74%. Similarly, efficient anoxic transformation of ibuprofen (75%) was observed after an
adaptation period of 340 d. Temperature (16–26  C) only had a slight effect on the removal
of CTL which increased in 4%.
ª 2010 Elsevier Ltd. All rights reserved.

1. Introduction conditions, which can be installed in different compartments

Conventional Sewage Treatment Plants (STPs) have been
designed to simultaneously eliminate organic matter and
nitrogen from urban wastewater by the implementation of
technologies that combine the existence of anoxic and aerobic
of the plant (e.g. activated sludge plants), or be sequentially
applied in one single reactor (e.g. sequential batch reactors).
Nowadays, removal mechanisms for conventional
contaminants under different redox conditions are under-
stood in detail and have been efficiently applied in most full-

* Corresponding author. Tel.: þ34 981 56 31 00; fax: þ34 981 52 80 50.
E-mail addresses: sonia.suarez@usc.es (S. Suarez), juan.lema@usc.es (J.M. Lema), francisco.omil@usc.es (F. Omil).
0043-1354/$ – see front matter ª 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.watres.2010.02.040
 water research 44 (2010) 3214–3224 3215

scale STPs, although not the same can be said for micro-
Table 1 – Concentration of PPCPs spiked to the reactor
pollutants. For the latter, the contribution of anoxic and feed (CFeed in mg LL1), CAS number, solid–water
aerobic conditions to their overall removal was usually not distribution coefficient (Kd in L kgL1) and Henry’s law
analyzed, since only influent and effluent concentrations of constant (H in mg mL3 air/mg mL3 wastewater).
micropollutants have been normally considered in the moni- Compound CFeed CAS log Kd Ha
toring of STPs (Clara et al., 2005; Göbel et al., 2007; Jones et al.,
Anti-depressants
2007; Joss et al., 2005). There are some exceptions, as for
Fluoxetine (FLX) 20 054910-89-3 0.7b,c 3.6  106
example kinetic experiments performed under aerobic,
Citalopram (CTL) 20 059729-33-8 2.0b,d 1.1  109
anoxic and anaerobic redox conditions (Joss et al., 2004), as
well as individual samplings carried out in the denitrification Estrogens
17b-Estradiol (E2) 10 50-28-2 2.8e 1.5  109
and nitrification tanks in full-scale STP (Andersen et al., 2003),
17a-Ethinylestradiol (EE2) 10 57-63-6 2.5f 3.3  1010
in order to analyze the behavior of estrogens (estrone: E1, 17b-
estradiol: E2 and 17a-ethinylestradiol: EE2) under different Anti-inflammatories
Ibuprofen (IBP) 10 15687-21-1 0.9f 6.1  106
redox conditions. It has also been shown that for EE2 the
Naproxen (NPX) 10 22204-53-1 1.1g 1.4  108
enrichment of activated sludge in nitrifying bacteria could
Diclofenac (DCF) 10 15307-86-5 1.2f 1.9  1010
enhance its transformation into metabolites devoid of estro-
Anti-epileptic
genic activity (Vader et al., 2000). Additionally, biodegradation
Carbamazepine (CBZ) 20 298-46-4 0.1f 4.4  109
of some other pharmaceuticals (ibuprofen: IBP, diclofenac:
DCF and clofibric acid) in oxic and anoxic biofilm reactors was Antibiotics
investigated by Zwiener et al. (2000). Trimethoprim (TMP) 20 738-70-5 2.3h 9.8  1013
Roxithromycin (ROX) 20 80214-83-1 2.2i 2.0  1029
Understanding the behavior of Pharmaceutical and
Sulfamethoxazole (SMX) 20 723-46-6 2.4h 2.6  1011
Personal Care Products (PPCPs) under different redox condi- Erythromycin (ERY) 20 114-07-8 2.2b 2.2  1027
tions is not only essential for achieving deeper knowledge of
Musks
the whole wastewater treatment process, but also in order to
Galaxolide (HHCB) 40 1222-05-5 3.3f 5.4  103
predict further pathways of those contaminants once released
Tonalide (AHTN) 40 1506-02-1 3.4f 5.1  103
into the environment (e.g. groundwater recharge, degradation Celestolide (ADBI) 40 13171-00-1 3.9j 7.3  101
in surface water, etc.). In fact, different metabolites could be
Tranquilizer
formed under aerobic and anoxic conditions, as previously
Diazepam (DZP) 20 439-14-5 1.3f 1.5  107
reported for other pollutants such as nonylphenol ethoxylate
surfactants (Goel et al., 2003) or the pharmaceutical residue a Syracuse Research Corporation (SRC).
b Jones et al. (2002).
phenazone (Greskowiak et al., 2006), indicating differences in
c Brooks et al. (2003).
degradation pathways in anoxic and aerobic processes. This
d Stuer-Lauridsen et al. (2000).
could explain why some pollutants are better removed under e Clara et al. (2004a,b).
anoxic conditions despite the common evidence that higher f Ternes et al. (2004).
oxidation potentials in aerobic environments should favor g Urase and Kikuta (2005).
their degradation. In this way, Drewes et al. (2001) reported h Göbel et al. (2005).
negligible removal of tri-iodinated benzene derivatives (X-ray i Joss et al. (2005).
j Kupper et al. (2006).
contrast media) under aerobic redox conditions, which was
significantly enhanced in anoxic environments.
The objective of the present work was to evaluate the
potential of nitrifying and denitrifying conditions for the Both reactors have been running at a Hydraulic Retention
elimination of a set of 16 Pharmaceutical and Personal Care Time (HRT) of 1 d.
Products (PPCPs), including pharmaceuticals from different The anoxic reactor was mechanically stirred and capped in
therapeutic classes, hormones and musk ingredients (Table 1), order to restrict the transfer of oxygen from air to the liquid
in order to better understand the overall removal process for phase. Recirculation of biomass from the settler to the reactor
such micropollutants in full-scale STPs. was carried out by means of a peristaltic pump in order to ach-
ieve an external recirculation ratio (Rext) of 30. Since the reactor
was aimed at promoting a denitrification process, a synthetic
feed containing an organic carbon source and nitrate was
2. Materials and methods continuously pumped into the system at a flow rate of
1.72  0.35 L d1 and with the following composition: 500 mg L1
2.1. Denitrifying and nitrifying reactors of COD (as NaCH3CO2), 40 mg L1 of N-NO3, 8 mg L1 of P-PO4 and
a solution of trace elements (FeCl3, H3BO3, CuSO4, KI, ZnSO4,
Two 2-L continuous stirred-tank reactors coupled to a 1-L CoCl2, MnCl2 at concentrations in the range of 3–150 mg L1).
settler were inoculated with biomass collected from In the aerobic plant oxygen was supplied through air
a conventional activated sludge pilot plant which was oper- diffusers located at the bottom of the reactor and recirculation
ated for more than 1.5 years in order to study the fate of the of biomass was carried out by means of an air lift device in
same set of PPCPs in a different reactor configuration. One order to maintain a Rext of 40. To achieve nitrifying condi-
reactor was operated under anoxic conditions, whereas in the tions, the reactor was fed at a flow rate of 1.94  0.27 L d1 with
second plant nitrifying aerobic conditions were maintained. an inorganic carbon source and ammonia at concentrations of
3216 water research 44 (2010) 3214–3224

1000 mg L1 NaHCO3, 40 mg L1 of N-NH4, 8 mg L1 of P-PO4
and the same trace elements as those employed in the anoxic
reactor.
A start-up period, during which no PPCPs were added to the
reactors, was maintained for at least one Solid Retention Time
(SRT) in order to allow acclimation of bacterial population to
the new redox and feeding conditions. After this initial stage,
pharmaceuticals were incorporated to the feed at the
concentrations indicated in Table 1. PPCPs were directly
spiked into the feeding tank from stock solutions of individual
compounds dissolved in methanol (antibiotics and anti-
inflammatories) or acetone (musks, anti-depressants, CBZ
and DZP) at a concentration of 2000 mg L1 which were stored
in the freezer. Both reactors have been continuously working
during 440 d at constant operation parameters, with two
exceptions. The first is temperature which varied seasonally
(16–26  C) since it was not controlled (Fig. 1), while the second
is SRT for which two operational periods were distinguished:
SRT >50 d and <50 d; SRT >20 d and <20 d for the nitrifying
and anoxic system, respectively. The variability of SRT is due
to variable losses of biomass within the effluent derived from
the use of a settler as separation unit, but also due to the
fluctuations of biomass concentration inside the reactors
which was especially important in the aerobic plant (Fig. 1). In
the latter, biomass concentration decreased down to
0.5 g VSS L1 until day 170, most probably due to the promo-
tion of endogenous respiration of the heterotrophic bacteria
A 2.5 30
2.0 25
VSS (g.L-1)
present in the inoculum, in the absence of an organic matter
source. After this initial stage, bacterial population stabilised
and initiated a growth period until day 300, at a rate of
7.8  103 g VSS d1, achieving a pseudo-steady sludge
concentration of 1.6 g VSS L1. The yield constant of produced
biomass per amount of ammonia oxidized was 0.1 g VSS g1
N-NH4, which is in accordance with the stoichiometric
parameter.
2.2. Analytical methods
Both reactors were weekly sampled, including the feed, reac-
tion medium and final effluent, in order to analyze conven-
tional parameters and PPCPs. Total and Volatile Suspended
Solids (TSS and VSS), nitrite and nitrate concentrations were
determined following Standard Methods (APHA, 1999),
ammoniacal nitrogen was measured with a spectrophotom-
eter (Shimadzu UV-1603, UV–visible) at 635 nm and Total,
Inorganic and Organic Carbon (TC, IC and TOC) were deter-
mined by a Shimadzu analyzer (TOC-5000). Additionally,
temperature, pH and dissolved oxygen have been regularly
followed in both reactors.
Analyses of PPCPs were performed after collecting 2-L
samples in glass or aluminum bottles, which were immediately
prefiltered (AP4004705, Millipore) and supplemented with
a pinch of sodium azide (w0.3 g L1). For the analysis of PPCPs
sample extraction based on Solid Phase Extraction (SPE) or Solid
Phase MicroExtraction (SPME) was used as pre-concentration
technique prior to their quantitative determination. Liquid or
Gas Chromatography coupled to Mass Spectrometry (LC–MS or
GC–MS, respectively) was used for the final quantification.
Analysis of the soluble content of anti-inflammatory
compounds, carbamazepine (CBZ), diazepam (DZP) and

1.5 20 musks was performed following the methodology described

T (ºC)

in Rodrı́guez et al. (2003), which consists of adjusting the pH of

1.0 15 the samples to 2.5, adding meclofenamic acid and dihy-
drocarbamazepine as surrogate standards, SPE of 250 mL
samples using 60 mg OASIS HLB cartridges (Waters, Milford,
0.5 10
MA, USA) and elution from the cartridge using 3 mL of ethyl
acetate. This extract was divided into two fractions: one of
0.0 5
0 100 200 300 400 them was used for direct determination of CBZ, DZP, galax-
olide (HHCB), tonalide (AHTN) and celestolide (ADBI), while
time (d)
the other one was employed for the analysis of anti-
B 2.5 30
inflammatories as their tertbutyldimethylsilyl derivatives.
Finally, GC/MS detection was carried out in a Varian CP 3900
2.0 27 chromatograph (Walnut Creek, CA, USA) equipped with
a split–splitless injector and connected to an ion-trap mass
VSS (g.L-1)

1.5 24 spectrometer. Additionally, total concentrations of musks

T (ºC)

were determined by Solid Phase Micro Extraction (SPME)

1.0 21 following the procedure developed by Garcı́a-Jares et al. (2002).
Briefly, 10-mL samples were immersed in a bath at 100  C for
0.5 18 5 min to equilibrate temperature. Then, a PDMS–DVB fiber
(65 mm polydimethylsiloxane–diviylbenzene, Supelco, USA)
0.0 15 was exposed to the headspace over the sample for 25 min.
0 100 200 300 400 Once the exposition finished, the fiber was immediately
inserted into the GC injector for chromatographic analysis.
time (d)
The anti-depressants fluoxetine (FLX) and citalopram (CTL)
Fig. 1 – Temperature (>), VSS inside the reactor (:) and in were analyzed by GC–MS (Varian Saturn 3) after SPME
the effluent (C) of the nitrifying (A) and denitrifying (B) according to Lamas et al. (2004). Briefly, water samples were
reactor. placed in 22-mL headspace vials. To improve the extraction
 water research 44 (2010) 3214–3224
a derivatization process was carried out with potassium
hydrogen carbonate and acetic anhydride (acetylation).
Afterwards SPME at 100  C was carried out using a PDMS-DVB
fiber which was inserted into the GC injection port after
30 min in order to complete the analysis.
Estrogens were analyzed according to Quintana et al.
(2004). Accordingly, the pH of the samples was adjusted to 6
and methanol (1%) and the internal standard, 17b-estradiol-d4
(75 ng L1), were added to the samples prior to its pre-
concentration by SPE (Oasis HLB 60 mg cartridges).
Cartridges were then dried and eluted with 3 mL of ethyl
acetate. This volume was reduced to approximately 0.3 mL
and further cleaned-up by passing it through a 500 mg Sep-
Pak silica cartridge. Analytes were then eluted with 10 mL of
ethyl acetate, and the extract reduced to 0.1 mL and derivat-
ized with N-methyl-N-(trimethylsilyl)trifluoroacetamide
(MSTFA) at 85  C for 100 min. GC–MS–MS analysis was carried
out using a Varian CP 3800 gas chromatograph connected to
ion-trap mass spectrometer (Varian Saturn 2000).
For analysis of antibiotics, sulfadimidin-C13 and Caffeine-
C13 were used as internal standards and an acidic buffer
solution was added to the samples prior to sample extraction.
SPE was carried out with Isolute 101 cartridges and analytes
were eluted with methanol and acidic methanol. Final detec-
tion was performed in an LC–MS–MS in the positive ESI mode.
2.3. Mass balances
Mass balances were applied considering stripping, sorption
and degradation as possible removal processes for PPCPs.
Volatilization was only considered in the mass balances of
the aerobic reactor, since the anoxic plant was not aerated.
The relative fraction stripped to the gas phase was calculated
according to Eq. (1):
Cj;air H$qair
¼ (1)
Cj;total 1 þ Kd;j $TSS þ H$qair
where C*j,air is the concentration of compound j that leaves the
reactor during aeration referred to the volume of wastewater
treated and Cj,total is the total concentration of compound j
(both in mg L1), H the dimensionless Henry’s law constant, qair
the aeration applied per unit of wastewater treated
(Lair L1wastewater), Kd,j the solid–water distribution coefficient
of compound j (L kg1) and TSS the suspended solids
concentration inside the reactor (kg L1).
According to Eq. (1) volatilization is not significant for any
compound (<5%, even for the relatively volatile compounds
galaxolide: HHCB and tonalide AHTN), except for celestolide
(58%), even under worst-case conditions (i.e. qair of
15 Lair L1wastewater and TSS inside the aerobic plant of 0.8 g L1).
For the latter, the mass flow that leaves the aerobic reactor due
to volatilization (Fj,Stripped in mg d1) has been calculated
according to Eq. (2) and included in the mass balances:
Fj;Stripped ¼ Cj;air $Q (2)
being Q in Eq. (2) the feed flow rate in the aerobic reactor
(L d1).
The mass flow of compound j leaving the reactors sorbed
onto solids contained in the final effluent (Fj,Sol in mg d1) has
3217
been estimated applying Eq. (3), where sorption equilibrium is
assumed:
Fj;Sol ¼ Q$Kd;j $TSSEff $Cj;Eff (3)
where, Cj,Eff is the dissolved concentration of compound j
(mg L1) and TSSEff the suspended solids concentration (kg L1),
both measured in the effluent.
Sorption coefficients (Kd,j) considered in the mass balances
have been taken from the literature (Table 1) with the excep-
tion of fragrances (HHCB, AHTN and celestolide: ADBI) for
which this parameter has been calculated from experimental
total and soluble concentrations measured in the effluent
(Ternes et al., 2004). Priority was always given to experimen-
tally determined values, although in the case of the two anti-
depressants (FLX and CTL) this parameter had to be estimated
from their KOW, following the procedure described in Jones
et al. (2002). It is worth to highlight that Kd values reported
by Göbel et al. (2005) for SMX vary up to 70%, being the reasons
for this not completely clear. Taking into account the physico-
chemical properties of this compound, sorption is not expec-
ted to be significant under the operational conditions
considered. On the one hand, lipophilic interactions with the
lipid fraction of the sludge (i.e. absorption) should not be
significant according to the log Kow of 0.9 reported for SMX
(Syracuse Research Corporation). Compounds can also sorb
onto sludge through the establishment of electrostatic inter-
actions between a positively charged compound and the
negatively charged surface of microorganisms (i.e. adsorp-
tion). According to Lin et al. (1997), SMX exhibits a positively
charged amino group at a very acidic pH (pKa1 1.8), while at pH
higher than 5.7 (pKa2) a loss of the sulfonamide proton yields
to its negatively charged conjugate, thus indicating that
interactions between SMX and sludge are not probable.
Total mass flow of compound j in the effluent (Fj,Eff in
mg L1) has been calculated as the sum of the flow in the liquid
and the solid phase (Eq. (4)).


Fj;Eff ¼ Cj;Eff $Q$ 1 þ Kd;j $TSSEff (4)
In the case of influents, the mass flow (Fj,Feed in mg L1) has
been calculated assuming that sorption is negligible, since the
synthetic feed did not contain any solid particles:
Fj;Feed ¼ Cj;Feed $Q (5)
where, Cj,Feed is the concentration of compound j in the feed
(mg L1).
Assuming steady-state conditions for the reactors, biolog-
ical transformation can be calculated according to Eq. (6):
Fj;Feed  Fj;Eff
Ej;Anox ¼ $100
Fj;Feed

ð6Þ
Fj;Feed  Fj;Eff þ Fj;Stripped
Ej;Aer ¼ $100
Fj;Feed
where, Ej,Anox and Ej,Aer are the transformation efficiencies (%)
for compound j in the anoxic and aerobic reactors, respectively.
2.4. Degradation kinetics
Biological transformation of PPCPs can be described by
pseudo-first order kinetics (Joss et al., 2006):
3218 water research 44 (2010) 3214–3224

dCj;total Ten of the considered PPCPs were removed to a high degree

rS ¼  ¼ kbiol $VSS$Cj;dissolved
dt
where Cj,total and Cj,dissolved are the total and dissolved
concentrations of compound j (mg L1), t the time (d), kbiol is the
reaction rate constant (L g1 d1) and VSS the volatile sus-
pended solids concentration (g L1).
For calculating the pseudo-first order degradation rate
constant (kbiol) for the selected PPCPs the processes were
adjusted to a steady-state, continuous stirred-tank reactor
model:

et al., 2006; Simonich et al., 2002), whereas eliminations
Fj;Feed  Fj;Eff þ Fj;Stripped  rS $V ¼ 0
where V is the reaction volume (L).
3. Results and discussion
3.1. Overall removal of PPCPs in the reactors
The efficiency of the aerobic reactor regarding nitrification
increased from w40% up to w90% during the first 30 d of
operation and remained afterwards stable around 98%. Simi-
larly, the anoxic reactor showed to be very efficient in the
removal of nitrate as nitrogen gas, reaching almost 100% after
the first 50 d of operation.
Mean concentrations of PPCPs in the influents and effluents
from both reactors together with their overall removal from the
liquid phase are summarized in Table 2. Data from the whole
operation period, without distinguishing between different
conditions of temperature and SRT, were considered in the
calculation of the mean, which led in some cases to significant
standard deviations. In all cases, removal was calculated from
measured influent concentrations, rather than from those spiked
to the feeding tank (Table 1). The differences between both could
be attributed to losses of the spiked compounds due to degra-
dation or sorption within the feeding system.
(7)
(>85%) in the aerobic reactor, comprising hormones (E1 þ E2
and EE2), the anti-inflammatory drugs IBP and naproxen
(NPX), the three musks (HHCB, AHTN, and ADBI), the anti-
depressant fluoxetine (FLX) and two antibiotics (roxi-
thromycin: ROX and erythromycin: ERY). These high removal
efficiencies occurring in aerobic treatment plants regarding
hormones, anti-inflammatory drugs and fragrances have
been already reported by several authors (Baronti et al., 2000;
Gomez et al., 2007; Joss et al., 2004; Kupper et al., 2006; Nakada
(8)
previously determined for FLX, ROX and ERY (Castiglioni et al.,
2006; Göbel et al., 2007; Joss et al., 2005; Vasskog et al., 2006)
were significantly lower than those measured in the present
work. The anoxic reactor has shown to be able to remove
fragrances, FLX and natural estrogens (E1 þ E2) in an effective
way, although in a slightly lower degree (>70%) compared to
the aerobic reactor, whereas for the rest of these ten
compounds removal achieved was much less effective (<40%).
Anti-depressant citalopram (CTL) has been partially
removed in both reactors (60% and 44% in the aerobic and
anoxic reactor, respectively), similar to the overall removal
efficiencies determined by Vasskog et al. (2006) during one
sampling campaign performed at three STPs in Norway.
The rest of pharmaceuticals (DCF, sulfamethoxazole: SMX,
diazepam: DZP, trimethoprim: TMP and carbamazepine: CBZ)
have not been significantly transformed (<25%) by the biolog-
ical treatment with neither nitrifying nor denitrifying bacteria.
The high persistence of these compounds has also been
observed in different full-scale STP (Clara et al., 2004a,b; Göbel
et al., 2007; Lindberg et al., 2005; Lindqvist et al., 2005), with the
exception of SMX for which removal efficiencies reported
varied in a wide range. For example, eliminations of 0–84% and
(-138)–60% can be found in Castiglioni et al. (2006) and Göbel
et al. (2007), respectively, although this could be partially due
to the fact that real wastewaters, which have a more complex

Table 2 – Mean concentrations of PPCPs and standard deviations (n [ 10) in the feed (CFeed) and effluent (CEff) of the aerobic
and anoxic reactors (ppb or mg LL1). Removal from the liquid phase (%) calculated for each time point, and afterwards
averaged over the whole operational period. LOD: Limit of Detection of the analytical method.
PPCP LOD (ng L1) Aerobic reactor Anoxic reactor

CFeed CEff Removal CFeed CEff Removal

E1 þ E2 0.7 6.6  1.4 0.07  0.04 99  0 6.4  0.8 1.8  0.3 72  2

IBP 27 8.1  1.5 0.4  0.3 95  4 8.0  0.7 5.1  2.0 37  26
HHCB 23 13.4  8.6 0.4  0.3 92  12 10.2  8.2 0.5  0.2 86  15
AHTN 23 15.5  11.0 0.7  0.3 90  13 11.7  9.8 0.9  0.1 82  16
ADBI 23 15.8  7.0 0.4  0.3 97  2 10.9  7.7 0.6  0.2 88  15
FLX 17 13.4  3.7 1.0  0.2 92  3 14.6  6.5 2.2  0.8 84  6
ROX 1.2 17.4  5.9 1.6  0.6 91  0 18.8  1.2 15.4  2.5 15  7
ERY 1.2 17.7  2.2 2.0  0.7 89  2 23.9  0.1 19.1  2.3 20  10
EE2 1.7 5.5  2.6 0.8  0.9 87  11 5.8  1.9 4.6  1.4 20  13
NPX 27 9.5  0.9 1.3  0.5 86  5 9.0  1.1 8.1  0.4 9  13
CTL 15 13.0  7.4 4.5  2.3 60  17 16.0  4.3 9.0  3.0 44  9
DCF 100 8.2  1.9 6.2  2.7 22  28 6.4  0.9 6.2  0.6 25
SMX 2.6 21.1  1.6 16.4  0.1 22  5 n.a. n.a. –
DZP 230 16.1  4.1 13.3  3.5 17  11 15.3  5.8 12.2  3.4 16  17
TMP 2.9 19.3  1.0 16.4  1.0 14  10 n.a. n.a. –
CBZ 470 19.0  4.9 18.1  5.9 6  12 17.9  4.8 17.9  5.6 1  10

n.a. not analyzed.

 water research 44 (2010) 3214–3224 3219

matrix, were used in these works. For the two antibiotics (SMX
and TMP) the behavior in the anoxic reactor could not be
established, since none of these compounds have been detec-
ted in the feed or effluent of the reactor, although both of them
had been spiked to the synthetic feed. The reason for that is not
completely clear, although interferences with other
compounds existing in the feed might be a plausible reason.
3.2. Fate of PPCPs in the reactor. Application of mass
balances
Mass balances were applied to each compound, according to
Eqs. (2)–(6) and results have been graphically represented,
including the contribution of both, biological transformation
(Ej,Anox and Ej,Aer) and sorption (Fj,sol/Fj,Feed  100) to the overall
removal of PPCPs. Moreover, volatilization has also been
considered in the case of ADBI in the aerobic plant (Fj,Stripped/
Fj,Feed  100). The residual fraction of each compound that
leaves the reactors with the effluent has been also included in
the plots (Cj,Eff  Q/Fj,Feed  100). The whole operation period of
both reactors has been classified according to temperature
(low: 16–20  C and high: 20–26  C) and SRT. Additionally,
pseudo-first order degradation rate constants, kbiol, were
calculated (Table 3) according to Eqs. (7) and (8) once stable
operational conditions were achieved and specific nitrifying
and denitrifying biomass was developed inside the reactors.
Faster degradation kinetics were measured in the nitrifying
reactor compared to the denitrifying system for the majority
of PPCPs, with the exception of fragrances for which very high
kbiol were determined in both reactors. According to their kbiol
the selected PPCPs could be classified as very highly
(kbiol > 5 L g1 1 1 1
SS d ), highly (1 < kbiol < 5 L gSS d ), moderately
(0.5 < kbiol < 1 L gSS d ) and hardly (kbiol < 0.5 L g1
1 1 1
SS d ) biode-
gradable, which in turn led to removal efficiencies of >80%,
60–80%, 40–60% and <40%, respectively (Table 3).
Diclofenac showed to be very persistent during treatment in
the anoxic reactor (kbiol < 0.04 L g1 1
SS d ), but this was not the
case inside the aerobic plant. For the latter, the data trend in
Fig. 2B seems to indicate that there has been an initial adap-
tation period that coincides with the death and washout of
heterotrophic bacteria (w170 d) during which removal of DCF
increased from 0% to 25%. Afterwards, a correlation between
sludge concentration in the reactor and biological trans-
formation of DCF was observed, reaching maximum removals
of around 74%. The high deviations observed in Fig. 2A for
aerobic conditions, indicate that the behavior of DCF was
influenced to a higher extent by the biomass developed in the
system than by the operation temperature and SRT. The fate of
DCF under anoxic and oxic conditions has been investigated by
Zwiener et al. (2000) in biofilm reactors who found removal
efficiencies below 20% under both conditions. Taking into
account that those biofilm reactors had been inoculated with
municipal sewage sludge and that operation stopped after only
120 d, these results are comparable to those obtained in the
present research during the first months.
Naproxen and FLX were both biologically transformed to
a high degree in the aerobic reactor (Fig. 3A and B, respec-
tively) with less than 16 and 9% of residual mass flow in the
effluent, respectively. While FLX exhibited significant trans-
formation in the anoxic treatment (79–89%), that was not the
case of NPX. Fluoxetine concentrations have been measured
in the influent and effluent of three STPs in Norway (Vasskog
et al., 2006) being the removal efficiencies reported in the
range of 8–70% for FLX, which are below the values measured
in the nitrifying reactor of the present study. This could be due
to the higher proportion of nitrifiers in this plant, although no
definite conclusion can be made since data for comparison are
very scarce. A positive effect of increasing the SRT of the
reactor has been observed for FLX in the anoxic reactor (kbiol
w5 and w2.5 L g1 SS d
1
at SRT >20 d and <20 d, respectively)

Table 3 – Summary of PPCP transformations obtained in the nitrifying and denitrifying reactors, biological pseudo-first
order reaction rate constants (kbiol in L gL1
SS d
L1
) estimated upon achievement of stable operational conditions and observed
influence of SRT, T and other sludge characteristics.
Compound Transformation kbiol (L g1 1
SS d ) Influence

Aerobic Anoxic Aerobic Anoxic SRT T Sludge

HHCB þþ þ/þþ 170 150 No No No

E1 þ E2 þþ þ 170 w3 Yes No No
AHTN þþ þ/þþ 115 60 No No No
ADBI þþ þ/þþ 75 75 No No No
IBP þþ /þ 20 w1.5 No No Yes
EE2 þþ / 20 0.4 Yes No No
FLX þþ þþ 9 w5 Yes No No
ROX þþ  9 0.2 n.a. n.a. No
ERY þþ / 6 0.15 n.a. n.a. No
CTL þ þ 3 0.5 Yes Yes No
NPX þþ  9 <0.2 Yes No No
DCF /þ  1.2 <0.04 No No Yes
SMX  n.a. 0.3 n.a. n.a. n.a. No
DZP   <0.4 <0.25 No No No
TMP  n.a. 0.15 n.a. n.a. n.a. No
CBZ   <0.06 <0.03 No No No

() <20%; () 20–40%; (þ) 40–60%; (þ) 60–80%; (þþ) >80%; n.a. not analyzed. The influence of SRT, temperature (T ) and other characteristics of
sludge (Sludge) on the transformation degree is indicated as (yes) or (no).
3220 water research 44 (2010) 3214–3224

A Aerobic Anoxic B
120% 70

100% t>400d

DCF removal (%)

50
DCF fate

80%
t: 170-340d
60% 30

40%
10 t<100d
20%

0 0.5 1 1.5 2 2.5

0% -10
SRT: <50d >50d >50d <20d >20d
VSS (g/L)
T: high high low high high

C Aerobic Anoxic D
2.5 100
120%

100% 2 80

IBP removal (%)

VSS (g/L)
80%
1.5 60
IBP fate

60%
1 40
40%

0.5 20
20%

0% 0 0
SRT: <50d >50d >50d <20d >20d <20d 0 100 200 300 400 500
T: high high low high high low
time (d)

Fig. 2 – A, C) Fate of DCF and IBP in the aerobic and anoxic reactors, indicating the contribution of biological transformation
( ), sorption (-) and release within the effluent (,). B) Correlation between removal of DCF and biomass concentration in
the aerobic reactor for the different sampling dates (t). D) Correlation between removal of IBP in the anoxic reactor (C) and its
biomass concentration (>).

and NPX in the aerobic plant, although for the latter the day 0 and 200; 0.3 L g1
SS d
1
from day 200 to 350 and 1.5 L g1
SS d
1

increase in removal was very slight (3%). At the operational
conditions of these reactors it is not expected that an increase
in the SRT will lead to changes in the bacterial population,
although, as described in Omil et al. (2010) the enzymes
produces by them could act in a different way. For example,
Jones et al. (2007) propose that systems operating at high SRT
could promote the release of less specific enzymes due cell
lysis. Acclimation phenomena are also possible to occur,
during which microorganisms present in a given system are
able to degrade at a larger extent certain pollutants after
a period of time due to the broadening of their enzymatic
spectrum as has been shown for different xenobiotic (Layton
et al., 2000; Zwiener et al., 2000).
Ibuprofen and NPX exhibit both low sorption potential and
high aerobic biological degradation constants (kbiol IBP:
w20 L g1 1 1 1
SS d ; kbiol NPX: w9 L gSS d ). As expected from their
kbiol, aerobic transformation of IBP was slightly better than for
NPX, between 93 and 96%. When the fate of IBP in the anoxic
process was analyzed according to operational conditions of
temperature and SRT, high deviations were observed (Fig. 2C),
thus, to better analyze its behavior, removal was plotted as
a function of time (Fig. 2D). The evolution of the biological
degradation rate constant with time (kbiol: 0.1 L g1
SS d
1
between
afterwards) indicates that adaptation of bacteria was respon-
sible for the wide range of transformation efficiencies
measured (16–75%). Adaptation of bacteria seems not to be
related to the presence of the pharmaceutical in the waste-
water, since biomass was taken from a reactor that had already
been fed with IBP for six months. Therefore, adaption was more
plausible due to changes in the characteristics of bacterial
population (e.g. its enzymatic spectrum) which developed
from a standard heterotrophic to a strict denitrifying biomass.
Zwiener et al. (2000) measured removals for IBP of more than
90% under oxic and of 15% under anoxic conditions, although,
as stated previously, they could have missed adaptation due to
a too early stop of the reactors (120 d).
Natural estrogens (E1 and E2) were highly transformed (99%)
under aerobic conditions and even in the anoxic reactor
transformation was significant (69–73%, Fig. 3C). Accordingly,
the calculated biological degradation constants were very high
in the aerobic and anoxic reactors, respectively (kbiol aerobic:
170 L g1
SS d
1
and kbiol anoxic: 3 L g1 1
SS d ). In the anoxic reactor,
a slight increase in the transformation degree was observed
when increasing the SRT of the plant (kbiol from 2.2 to
2.7 L g1 1
SS d ). Important eliminations of natural estrogens
under denitrifying conditions in full-scale STPs have also been
 water research 44 (2010) 3214–3224 3221

Aerobic Anoxic Aerobic Anoxic

A 120% B 120%
100% 100%

80% 80%
NPX fate

FLX fate
60% 60%

40% 40%

20% 20%

0% 0%
SRT: <50d >50d >50d <20d >20d SRT: <50d >50d >50d <20d >20d <20d
T: high high low high high T: high high low high high low

C Aerobic Anoxic D Aerobic Anoxic

120% 120%

100% 100%

80% 80%
E2+E1 fate

EE2 fate
60% 60%

40% 40%

20% 20%

0% 0%
SRT: <50d >50d >50d <20d >20d SRT: <50d >50d >50d <20d >20d
T: high high low high high T: high high low high high

Fig. 3 – Fate of NPX, FLX, natural hormones (E1 D E2) and EE2 in the aerobic and anoxic reactors, indicating the contribution
of biological transformation ( ), sorption (-) and release within the effluent (,).

reported by Andersen et al. (2003). The synthetic hormone
ethinylestradiol was only transformed appreciably in the
aerobic reactor (82–90%), whereas under anoxic conditions less
than 26% of the parent compound was degraded (Fig. 3D). This
behavior fits very well with the kinetic behavior of EE2 deter-
mined by Joss et al. (2004) for different redox conditions, where
it was shown that EE2 was removed at a significant rate only
under aerobic conditions. This was additionally observed in
combined anoxic/aerobic treatment plants (Andersen et al.,
2003). The role of ammonium monooxygenase (AMO), which
is the enzyme that catalyses the first step in nitrification, in the
degradation of EE2 was studied by different authors (Forrez
et al., 2008; Shi et al., 2004; Vader et al., 2000; Yi and Harper,
2007). Nitrifying sludge was reported to enhance trans-
formation of EE2, via hydroxylation that converts EE2 into
hydrophilic products devoid of estrogenic activity (Vader et al.,
2000), which was confirmed in the present work by comparing
kbiol obtained before the washout of heterotrophic bacteria
from the aerobic reactor and after the development of nitri-
fying sludge (kbiol: 9 and 20 L g1 1
SS d , respectively). This was
additionally supported by Shi et al. (2004), who studied the role
of ammonia oxidizing bacteria on the degradation rate
constants of these estrogens, observing that when they are
inhibited, degradation of estrogens still occurs but at a slower
rate, being this specially significant for EE2. Yi and Harper
(2007) determined a linear relationship between nitrification
and EE2 removal in enriched nitrifying cultures. The biological
degradation rate constant estimated for heterotrophic sludge
fits well with that reported by Joss et al. (2004) concerning
aerobic batch experiments with sludge from a conventional
activated sludge treatment plant (kbiol: 8  2 L g1 1
SS d ). The
transformation efficiency for EE2 in the aerobic reactor
increased around 8% when the plant was operated at higher
SRT.
In the case of antibiotics only two sampling campaigns
have been carried out, both at the same conditions of low
temperature and SRT below 20 d (Fig. 4). Roxithromycin and
erythromycin were transformed very efficiently (w90%) in the
aerobic reactor and to a larger extent than at similar SRTs in
full-scale STP (Göbel et al., 2007). This might point to a higher
affinity of nitrifying bacteria towards these compounds,
especially when comparing kbiol obtained in the present work
(kbiol ROX: 9 L g1
SS d
1
and kbiol ERY: 6 L g1 1
SS d ) with those
determined for activated sludge in Joss et al. (2006). On the
other hand, only slight transformations of these two antibi-
otics have been observed in the anoxic reactor (<27%). A slight
preference for a metabolism under oxic conditions for ROX
was also reported by Heberer et al. (2008) when studying the
influence of redox conditions on the elimination of antimi-
crobial residues during bank filtration. The other two antibi-
otics considered (SMX and TMP) have shown higher
persistence towards aerobic biological treatment, since the
maximum transformation observed was 26% and 21% for SMX
and TMP, respectively.
3222 water research 44 (2010) 3214–3224

Aerobic Anoxic available in the literature. The efficiency of the anoxic plant
120% was somewhat lower, although still quite significant (41–46%).
For musk compound ADBI the three removal mechanisms,
100% i.e. volatilization, sorption and biodegradation, have to be
Antibiotics fate

80%
60%
40%
20%
0%
ROX ERY- SMX
H2O
Fig. 4 – Fate of antibiotics (ROX, ERY, SMX and TMP) in the
aerobic and anoxic reactors, indicating the contribution of
biological transformation ( ), sorption (-) and release
within the effluent (,).
Transformation of CTL in the aerobic reactor increased
from 62% up to 70% when SRT was raised to above 50 d and it
also improved a 4% when operating at higher temperatures.
When comparing this data with those included in Vasskog
et al. (2006), again an increase in removal efficiencies seems
to be achieved when enriching activated sludge in nitrifying
bacteria, although, as stated for Fluoxetine, this should be
confirmed with more data about CTL which are currently not
considered in the mass balance applied to the aerobic reactor
(Fig. 5), whereas for the other two fragrances (HHCB and
AHTN) volatilization could be neglected. Sorption coefficients
(Kd) applied in Eq. (4) have been experimentally determined
from measured soluble and total concentrations of fragrances.
The following results were obtained: 1.5  103 and
4.9  103 L kg1 for HHCB, 2.0  103 and 3.4  103 L kg1 for
AHTN and 5.2  102 and 3.9  103 L kg1 for ADBI, in the
TMP ROX ERY- aerobic and anoxic reactor, respectively. These values are in
H2O
the range of 2  103–1  104 L kg1 previously reported for
activated sludges (Joss et al., 2005; Kupper et al., 2006; Ternes
et al., 2004), except for ADBI in the aerobic plant where the
measured sorption coefficient was somewhat lower. For these
three compounds, sorption coefficients determined for the
anoxic reactor were somewhat higher than those obtained for
the aerobic unit, which is a factor commonly not considered
when applying mass balances in full-scale STP. Therefore, it is
strongly recommended to measure those coefficients for each
particular situation, especially in the case of highly lipophilic
compounds, at least until the significant discrepancies
between published Kd values and environmental factors that
can affect this partition coefficient are better understood.
Transformation of HHCB and AHTN during aerobic bio-
logical treatment reached 79–99% and 76–98%, respectively,

Aerobic Anoxic Aerobic Anoxic

120% 120%

100% 100%

80% 80%
AHTN fate
HHCB fate

60% 60%

40% 40%

20% 20%

0% 0%
SRT: <50d >50d >50d <20d >20d <20d SRT: <50d >50d >50d <20d >20d <20d
T: high high low high high low T: high high low high high low

Aerobic Anoxic
120%

100%

80%
ADBI fate

60%

40%

20%

0%
SRT: <50d >50d >50d <20d >20d <20d
T: high high low high high low

Fig. 5 – Fate of HHCB, AHTN and ADBI in the aerobic and anoxic reactors, indicating the contribution of biological
transformation ( ), sorption (-), release within the effluent (,) and for ADBI volatilization in the aerobic reactor ( ).
 water research 44 (2010) 3214–3224
whereas in the anoxic plant the efficiency was slightly lower,
in the range of 67–84% and 65–76%, respectively. The fraction
of these substances that left the reactors sorbed onto the
solids contained in the effluent was negligible in the aerobic
reactor and below 18% in the anoxic one (Fig. 5). Residual
concentration of ADBI in the aerobic effluent was below 4%,
being the two most significant removal pathways biological
transformation (80–96%) and volatilization (3–16%), since
contribution of sorption was negligible (Fig. 5). The anoxic
reactor also demonstrated high efficiency in the overall
removal of ADBI (78–93%), although in this case sorption (7–
10%) and biodegradation (69–89%) were the responsible
removal processes (Fig. 4). The very high biological degrada-
tion rate constants determined for fragrances (kbiol aerobic:
>75 L g1 1
and kbiol anoxic: >60 L g1 1
SS d SS d ) does not corre-
spond with the behavior of fragrances described in previous
works, where their removal was associated to sorption rather
than to biological transformation (Bester, 2004; Joss et al.,
2005; Kupper et al., 2006). These differences could be a conse-
quence of working with higher SRT in the present reactors
compared to the mentioned works, where SRT below 25 d
were considered. This assumption is based on the consider-
ation that for lipophilic compounds, such as fragrances, the
retention time inside the reactor is determined by the SRT,
rather than by the HRT of the plant. Similar biotransformation
percentages to the measured in this study have been reported
by Clara et al. (2005) for AHTN and HHCB in a STP working at
SRT >52 d, which supports the previous discussion.
4. Conclusions
Considering the main removal mechanisms for PPCPs only
(bio)transformation was significant for the majority of
compounds. In the case of musk fragrances, a significant
fraction (7–18%) of compounds left the anoxic reactor sorbed
onto solids, whereas sorption was negligible in the case of the
aerobic plant, which was associated to the better settling
characteristics of the nitrifying biomass developed in that
reactor. Volatilisation was only significant for ADBI and
contributed between 3 and 16% to the removal of this
substance in the aerobic system.
The selected PPCPs could be classified according to their
aerobic and anoxic biodegradability as follows:
 Highly biodegradable under aerobic and anoxic conditions:
IBP, FLX, natural estrogens (E1 þ E2) and musk fragrances
(HHCB, AHTN and ADBI).
 Highly biodegradable under aerobic conditions but persis-
tent in the anoxic reactor: DCF, NPX, EE2, ROX and ERY.
 Moderately biodegradable under aerobic and anoxic condi-
tions: CTL.
 Resistant to biological transformation: SMX, TMP, CBZ
and DZP.
The positive effect of increasing SRT has been demon-
strated for several compounds, with improvements in the
removal efficiency of w10% in the case of FLX, CTL and EE2,
whereas temperature only affected very slightly the removal
of CTL (4%). The characteristics of bacteria developed in the
3223
reactors influenced very significantly the fate of DCF and IBP
in the plants. In fact, removal of those compounds was only
achieved after the growth of specific bacteria. The enrichment
of the activated sludge used as inoculums in nitrifying
bacteria has shown to enhance transformation of EE2, ROX
and ERY, which are compounds moderately transformed in
conventional activated sludge plants.
Acknowledgments
This work was supported by Spanish Ministry of Education
and Science (MICROFARM project: CTQ2007-66265/PPQ,
NOVEDAR_Consolider project: CSD2007-00055 and Research
Fellowship). The authors thank Oliver Gans (Austrian Federal
Environment Agency) for the analyses of antibiotics.
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