Environment International 69 (2014) 2839

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Environment International
journal homepage: www.elsevier.com/locate/envint

Review

Fate of diclofenac in municipal wastewater treatment plant A review

Niina Vieno a,, Mika Sillanp b,1
a
Envieno, Logomo Byr, Kydenpunojankatu 14, FI-20100 Turku, Finland
b
Lappeenranta University of Technology, Laboratory of Green Chemistry, Innovation Centre for Safety and Material Technology, Sammonkatu 12, FI-50130 Mikkeli, Finland

a r t i c l e i n f o a b s t r a c t

Article history: Diclofenac (DCF) is a common anti-inammatory pharmaceutical that is often detected in waste wasters,
Received 12 November 2013 efuents and surface waters. Recently, DCF was included in the watch list of substances in EU that requires
Accepted 20 March 2014 its environmental monitoring in the member states. DCF is also known to harmfully affect several environ-
Available online xxxx
mental species already at concentrations of 1 g/l. This review focuses on the occurrence and fate of DCF
in conventional wastewater treatment processes. Research done in this area was gathered and analyzed in
Keywords:
Pharmaceutical
order to nd out the possibilities to enhance DCF elimination during biological wastewater treatment. More
Biotransformation precisely, human metabolism, concentrations in wastewater inuents and efuents, elimination rates in the
Sorption treatment train, roles of sorption and biotransformation mechanisms during the treatment as well as for-
Conventional activated sludge mation of transformation products are reported. Additionally, the effect of process conguration, i.e. con-
Membrane bioreactor ventional activated sludge (CAS), biological nutrient removal (BNR), membrane bioreactor (MBR) and
Attached-growth bioreactor attached-growth bioreactor, and process parameters, i.e. solids retention time (SRT) and hydraulic reten-
tion time (HRT) are presented. Generally, DCF is poorly biodegradable which often translates into low elim-
ination rates during biological wastewater treatment. Only a minor portion is sorbed to sludge. MBR and
attached-growth bioreactors may result in higher elimination of DCF over CAS or BNR. Long SRTs
(N 150 d) favor the DCF elimination due to sludge adaptation. Longer HRTs (N 23 d) could signicantly in-
crease the elimination of DCF during biological wastewater treatment. Bioaugmentation could be used to
enhance DCF elimination, however, this requires more research on microbial communities that are able
to degrade DCF. Also, further research is needed to gain more information about the deconjugation process-
es and biotic and abiotic transformation and the nature of transformation products.
2014 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
1.1. Human metabolism of DCF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
1.2. Occurrence of DCF in wastewaters, efuents, environment and drinking waters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
1.3. Ecotoxicological effects of DCF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2. Sorption of DCF to sewage sludge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
2.1. Removal of DCF in sludge treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3. Biological elimination and transformation of DCF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.1. Biotransformation mechanism of DCF . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
3.2. Biotic and abiotic transformation products . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
4. Elimination of DCF during biological wastewater treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
4.1. Effect of process conguration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
4.2. Effect of process parameters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
4.2.1. Solids retention time (SRT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36
4.2.2. Hydraulic retention time (HRT) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

Corresponding author. Tel.: +358 50 5448431.

E-mail addresses: niina.vieno@envieno.com (N. Vieno), mika.sillanpaa@lut. (M. Sillanp).
1
Tel.: +358 400 205215.

http://dx.doi.org/10.1016/j.envint.2014.03.021
0160-4120/ 2014 Elsevier Ltd. All rights reserved.
 N. Vieno, M. Sillanp / Environment International 69 (2014) 2839
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Introduction
Diclofenac (2-(2-(2,6-dichlorophenylamino)phenyl)acetic acid)
(DCF) is a common non-steroidal anti-inammatory drug (NSAID)
that is used as oral tablets or as a topical gel. It is sold under the com-
mercial names of Acoam, Algosenac, Almiral, Ana-Flex, Anthraxiton,
Antiam, Arcanafenac, Arthrex, Arthrifen, Arthtotec, Diclabeta, Diclac,
Dicloabac, Diclodoc, DiclofenacRatiopharm, Diclofenbeta, Diclomex,
Diclowal, Dicuno, Difen, Diklotab, DolgitDiclo, Eese, Effekton, Jutafenac,
Monoam, Motifene Dual, Rewodina, Sigafenac and Voltaren. Its physic-
chemical properties are presented in Table 1 and molecular structure
in Fig. 1.
In the point of view of environmental regulations in EU, pharma-
ceuticals and hormones are a highly topical group of compounds. A
proposal was made during the revision of the Water Framework Di-
rective (2000/60/EC) in European Union that would have classied
DCF along with two estrogenic hormones as priority substances. In
the end, in Directive 2013/39/EU, DCF and the hormones were in-
cluded in the watch list of substances that will be established along-
side the list of priority substances (EU, 2013). Substances in the
watch list shall be monitored by the EU member states in their sur-
face waters for a maximum of four years. However, no environmen-
tal quality standards (EQS) were assigned for the watch list
substances. However, during the revision process, EQS value of
100 ng/l for inland waters and 10 ng/l for coastal water were pro-
posed for DCF. Due to the wide interest of regulators and the public
to DCF, this review focuses solely on this common anti-
inammatory drug. Its fate in the human body and during the munic-
ipal wastewater treatment is reviewed. Additionally, mechanisms of
sorption and biotransformation as well as formation of transforma-
tion products are discussed. The effect of process conguration, i.e.
conventional activated sludge (CAS), biological nutrient removal
(BNR), membrane bioreactor (MBR) and attached-growth bioreactor
as well as process parameters, i.e. solids retention time (SRT) and hy-
draulic retention time (HRT) are reviewed. No review has been pub-
lished that would concentrate on DCF, its fate and transformation
processes in conventional wastewater treatment and would have
aimed to identify the possibilities to increase its elimination by en-
hancing the existing treatment processes. Previously published re-
views have focused in reporting the concentrations of wide range
of micropollutants (Ratola et al., 2012) or their removals in various
biological systems (Li et al., 2014; Onesios et al., 2009; Verlicchi
et al., 2012).
Table 1
Physico-chemical properties of diclofenac.
Parameter Value Reference
Chemical formula C14H10Cl2NO2
CAS no 15307-86-5
15307-79-6 (disodium salt)
Water solubility 2.37 mg/L SRC (2013)
pKa 4.15 SRC (2013)
logKow 4.51 SRC (2013)
logKd,primary sludge 2.7 Ternes et al. (2004a)
2.3 Radjenovic et al. (2009)
logKd,secondary sludge 1.2 Ternes et al. (2004a)
2.1 Radjenovic et al. (2009)
logKd, MBR 2.32.5 Radjenovic et al. (2009)
logKd,digested sludge 1.32.2 Carballa et al. (2008)
29
37
37
1.1. Human metabolism of DCF
DCF is administered topically or orally and undergoes almost com-
plete biotransformation in the human body. Topical gel adsorption
was found to be 67% (Davies and Anderson, 1997). The remaining
part is either washed off the skin or is attached to clothing. Of the orally
administered dose, between 65 and 70% is excreted in urine and 2030%
in feces as the parent drug or as metabolites (Davies and Anderson,
1997; Stierlin and Faigle, 1979). The majority of DCF is metabolized in
the human body and only b 1% of the orally administered dose is excret-
ed as un-metabolized DCF. As a result of phase II metabolism involving
glucuronic acid and taurine, glucuronide and sulfate conjugates of DCF
are formed. These conjugates make up to 11% of the administered
dose (Davies and Anderson, 1997; Stierlin and Faigle, 1979). The
World Health Organization has dened a daily dose for diclofenac of
100 mg. Of this dose, less than 1 mg is eliminated from the human
body as DCF and about 11 mg as DCF conjugates. The rest of the admin-
istered dose is excreted as metabolites of DCF or their conjugates.
Metabolic pathway of DCF in human body is presented in Fig. 1. Six
phase I metabolites of DCF have been detected in human plasma,
urine and/or feces. According to Davies and Anderson (1997), the pat-
tern of DCF metabolites in human urine is the same after topical and
oral administration. In total, the six metabolites of DCF and the conju-
gates of these account for 90% and 65% of the total administered DCF
in urine and in feces, respectively (Blum et al., 1996; Davies and
Anderson, 1997; Faigle et al., 1988; Stierlin and Faigle, 1979). The
main human metabolites of DCF are 4-OH-DCF and 5-OH-DCF. Both of
them are excreted mainly in conjugated form and only less than 1% is
excreted unchanged (Stierlin and Faigle, 1979). Important metabolites
are also 3-hydroxy-DCF and 4,5-dihydroxy-DCF. The two remaining
metabolites, 3-OH-4-OCH3-DCF and 4-OH-3-OCH3-DCF are excreted
in urine only in trace amounts (Blum et al., 1996; Faigle et al., 1988).
In some animal models, 4-OH-DCF has been shown to have 30% of the
anti-inammatory and antipyretic activity of DCF (Davies and Anderson,
1997). However, according to Wiesenberg-Boettcher et al., the anti-
inammatory activity of the main metabolites of DCF is at least 10 times
lower compared to DCF activity (Wiesenberg-Boettcher et al., 1991).
1.2. Occurrence of DCF in wastewaters, efuents, environment and drinking
waters
In reviewed studies, the measured maximum concentrations of DCF
in municipal wastewaters vary between 0.44 and 7.1 g/l and the mean
concentrations are between 0.11 and 2.3 g/l (Table 2). According to
Sim et al. (2011), the maximum concentrations in hospital wastewater
reached 6.88 g/l and in pharmaceutical manufacturer's wastewater
203 g/l in South Korea. The values are signicantly higher than normal-
ly detected in municipal wastewater. On the other hand, Zorita et al.
(2009) measured similar concentrations (around 0.2 g/l) in both hos-
pital and municipal wastewater in Sweden. Municipal wastewater con-
centrations reect the consumption of DCF by the residents in the
particular sewer system. The consumption rates vary greatly between
countries and also within countries. This makes it difcult to determine
typical wastewater concentrations. The yearly consumption of DCF
has been reported to vary between 195 and 940 mg per inhabitant in
different countries (Carballa, 2005; Clara et al., 2005b; Finnish
Medicines Agency, 2013; Khan and Ongerth, 2004; Sakshaug, 2012;
Ternes, 1998).
In the efuents of municipal wastewater treatment plants, DCF is
among the most frequently detected pharmaceuticals (Verlicchi et al.,
30 N. Vieno, M. Sillanp / Environment International 69 (2014) 2839

3-hydroxy-4-methoxy-diclofenac
4-hydroxy-3-methoxy-diclofenac
(3-OH-4-OCH3-DCF)
(3-OH-4-OCH3-DCF)

Diclofenac (DCF)

Diclofenac-1--o-acyl glucuronide
(DCF-G)

4-hydroxy-diclofenac 3-hydroxy-diclofenac 4,5-dihydroxy-diclofenac

5-hydroxy-diclofenac
(4-OH-DCF) (3-OH-DCF) (4,5-OH-DCF)
(5-OH-DCF)

R, R, R= not definitely identified ligands, presumably one per molecule

Fig. 1. Metabolic pathway of diclofenac (DCF) in human body.

Adapted from Blum et al. (1996), Davies and Anderson (1997), Faigle et al. (1988) and Stierlin and Faigle (1979).

2012). Due to its incomplete elimination during the treatment, efuent
concentrations rarely fall below the detection limits of few nanograms
per liter when analyzed using LCMS/MS or GC/MS. According to
Table 2, the efuent maximum concentrations vary between 0.12 and
4.7 g/l and mean concentrations between b 0.002 and 2.5 g/l. Accord-
ing to Verlicchi et al. (2012), out of 73 pharmaceuticals reviewed, DCF
had the eighth highest average mass load (240 mg/1000 inh) in the sec-
ondary efuent of municipal wastewater treatment plants. In addition,
the metabolites of DCF can also enter the environment via WWTP efu-
ents. Langford and Thomas (2011) reported that in Norway, 5-OH-DCF
was measured up to a concentration of 3.7 g/l in WWTP efuents.
Reported surface water concentrations of DCF generally fall below
100 ng/l (Hernando et al., 2006; Hilton and Thomas, 2003; Kim et al.,
2007; Lin et al., 2005; Rabiet et al., 2006; Vieno, 2007). Some studies re-
port higher, but still generally lower than 500 ng/l, surface water con-
centrations (Bendz et al., 2005; Buser et al., 1998; Kosjek et al., 2005;
Metcalfe et al., 2003; llers et al., 2001). Ternes (1998) reported a max-
imum concentration of 1200 ng/l in German rivers receiving sewage ef-
uents. In Karachi, Pakistan, a concentration of 8500 ng/l of DCF was
measured in Korangi drain that receives untreated residential and in-
dustrial efuents (Scheurell et al., 2009). Also, the hydroxylated
human metabolites, 3-OH-DCF and 4-OH-DCF, were detected in the
sample at concentrations of 300 and 1800 ng/l, respectively.
Generally, groundwater concentrations have been low or under
the detection limits (Lin et al., 2005; Loos et al., 2010; Lpez-Serna
et al., 2013; Rabiet et al., 2006). Few studies report higher groundwa-
ter concentrations. In the groundwater of Barcelona, diclofenac was
detected up to a concentration of 380 ng/l (Lpez-Serna et al.,
2013). Also, 4-OH-DCF was found up to a concentration of 147 ng/l.
Similar DCF concentrations (maximum of 380 ng/l) have also been
measured in groundwaters in Germany (Heberer, 2002). In drinking
waters, DCF concentrations have been reported as below or just
above the detection limits (17 ng/l) (Benotti et al., 2009;
Hernando et al., 2006; Kosjek et al., 2005; Lin et al., 2005; Vieno,
2007; Vulliet et al., 2011).
1.3. Ecotoxicological effects of DCF
Haap et al. (2008) reviewed literature studies that have evaluated
the ecotoxicology of DCF. They concluded that depending on the spe-
cies, exposure duration and endpoint used, effect concentrations varied
between 1 g/l and 80 mg/l. Generally, median effective concentrations,
i.e. EC50 values, using acute toxicity tests for Daphnia magna varied be-
tween 22 and 80 mg/l. Thus, a risk assessment based on routine tests
using for example D. magna mortality, would likely underestimate the
environmental toxicity of DCF. EC50 values are far higher than the envi-
ronment concentrations reported for DCF. Also chronic toxicity tests
(exposure time of 48 h10 days) using mortality, reproduction and em-
bryo and larvae mortality was reported to result in the lowest observed
effect concentrations (LOECs) of N2000 g/l for DCF (Ferrari et al., 2003).
Based on these studies, Ferrari et al. (2003) calculated a predicted no ef-
fect concentration (PNEC) for DCF as 116 g/l, i.e. 1000-fold higher than
are normally measured in the environment.
Table 3 summarizes studies that have shown ecotoxicological effects
of DCF at environmentally relevant concentrations using different bio-
markers as endpoints for exposure. Triebskorn et al. (2004) reported a
LOEC of 1 g/l for rainbow trout (Oncorhynchus mykiss) using changes
in liver ultrastructure, liver glycogen and kidney protein as the end-
point. Triebskorn et al. (2007) concluded that DCF showed more effects
on sh liver, kidney and gills than carbamazepine, clobric acid and
metoprolol. LOECs for damages in liver, kidney and gills were all at
1 g/l. Already extremely low DCF concentrations, i.e. 10 ng/l, was re-
ported to impair osmoregulatory ability of a green shore crab Carcinus
maenas (Eades and Waring, 2010). The authors suggested that this
caused the increase in the haemolymph osmolality recorded for the
crab. Lipid peroxidation was used as a biomarker for oxidative stress
in the studies of Quinn et al. (2011) and Feito et al. (2012). Quinn
et al. (2011) recorded signicantly increased levels of lipid peroxida-
tion, when zebra mussels (Dreissena polymorpha) were exposed to a
concentration of 1 g/l of DCF. Feito et al. (2012), on the other hand, re-
ported a reduction of lipid peroxidation in zebrash embryos already at
 N. Vieno, M. Sillanp / Environment International 69 (2014) 2839 31

Table 2
Concentrations of pharmaceuticals in municipal sewage treatment plant inuents and efuents in different countries.

Country Inuent (g/l) Efuent (g/l) Reference

Min Max Mean Median Min Max Mean Median

Austria 0.91 4.1 0.78 3.5 Clara et al. (2005a)

Canada 0.005 0.36 Metcalfe et al. (2003)
China 0.11 0.44 0.29 0.28 0.035 0.46 0.19 0.18 Sui et al. (2011)
China 0.03a 0.03a Yan et al. (2014)
China 0.28 0.34 0.32 0.12 0.17 0.15 Duan et al. (2013)
Europeb 5.5 0.68 Andreozzi et al. (2003)
Europec 0.15 1.4 Hernando et al. (2006)
Europed 1.5 0.29 Paxus (2004)
Finland 0.23 0.64 0.42 0.46 0.14 0.62 0.32 0.35 Vieno (2007)
France 0.21 0.49 Rabiet et al. (2006)
Germany 7.1 3.02 4.7 2.5 Heberer, 2002
Germany 2.10 0.81 Ternes (1998)
Germany 2.3e 1.6e Quintana and Reemtsma (2004)
Greece 0.86 2.17 0.15 1.1 0.41 Samaras et al. (2013)
Greece n.d. 5.2 0.28 n.d. 0.38 0.11 Kosma et al. (2014)
Pakistan 2.3e Scheurell et al., 2009
South Korea 0.13 0.04 Kim et al. (2007)
South Korea 0.09 0.52 0.24 0.193 0.05 1.8 0.19 0.09 Sim et al. (2011)
South Korea 0.08 n.d. Nam et al. (2014)
Spain 0.20 3.60 0.14 2.2 Gmez et al. (2007)
Spain 1.2 Kuster et al. (2008)
Spain b0.14 0.74 0.53 0.21 0.62 0.34 Gracia-Lor et al. (2012)
Spain b0.03 0.72 b0.015 0.74 Martn et al. (2012)
Spain n.d. 0.56 0.23 0.006 0.43 0.22 Rosal et al. (2010)
Spain/Croatia 0.50 0.25 0.50 0.32 Petrovic et al. (2006)
e
Sweden 0.16 0.12e Bendz et al. (2005)
Sweden 0.23 0.49 Zorita et al. (2009)
Switzerland 4.70 1.02 0.31 0.93 Buser et al. (1998)
Switzerland 0.10 0.70 llers et al. (2001)
Switzerlanda 0.10 0.53 0.05 0.56 Soulet et al. (2002)
Taiwan b0.002 Lin et al. (2005)
UK 0.41 0.46 Hilton and Thomas (2003)
e
USA 0.11 0.09e Yu et al. (2006)
USA 0.09 0.58 0.28 0.25 n.d. 0.12 0.012 n.d. Yu et al. (2013)

n.d. = not detected, b = value is below the detection limit.

a
Estimated from a gure.
b
Data from France, Greece, Italy and Sweden.
c
Data from Spain, Belgium, Germany and Slovenia.
d
Data from France, Greece, Italy, Sweden and Denmark.
e
n = 1.

a concentration of 0.03 g/l and an exposure time of 90 min. They also
reported phytotoxicological effects of DCF. An increase in mitochondrial
activity was reported for fern Polystichum setiferum after a 48-h expo-
sure to a DCF concentration of 0.3 g/l. Chronic effects for DNA of fern
were noted already at a concentration of 0.03 g/l. A study by Ericson
et al. (2010) showed that already at a concentration of 1 g/l, DCF affect-
ed negatively the byssus strength of the Baltic Sea blue mussel (Mytilus
edulis trossulus). The effect was more pronounced at higher DCF concen-
trations. This implies that the mussels impacted by DCF had reduced
ability to attach to the underlying substrate.
DCF may cause damages in sh eggs or embryos. Hallare et al. (2004)
reported that when zebrash (Danio rerio) embryos were exposed to
concentrations of b1000 g/l of DCF, no signicant embryotoxic or
proteotoxic effects were noted. However, according to Nassef et al.
(2010), DCF can cause damages in sh eggs or embryos if maternally
transferred into the eggs. They injected DCF into the eggs of Japanese
medaka sh (Oryzias latipes). At an exposure concentration of 12 ng/
egg, survival rate of the embryos was decreased to 67%, abnormal eye
development was reported in 9%, hemorrhage in 16%, yolk-sac shrink-
age in 11% and delayed development in 24% of the embryos. Signicant-
ly prolonged hatching times and the number of larvae failed to swim
upward was already noted at a concentration of 5 ng/egg compared to
control.
In addition to harmful effects to environmental organisms, DCF has
been shown to bioconcentrate in mussels and sh. Ericson et al.
(2010) measured signicantly higher concentrations of DCF in mussels
compared to water. At an environmental concentration of 1 g/l and ex-
posure duration of 8 d the bioconcentration factor was recorded to be in

Table 3
Studies showing ecotoxicological effects (LOEC = lowest observed effect concentration) of DCF on several species at environmentally relevant concentrations.

Species Exposure time Endpoint LOEC Reference

Oncorhynchus mykiss 28 d Changes in liver ultrastructure, liver glycogen and kidney protein 1 g/l Triebskorn et al. (2004)
Carcinus maenas nr Haemolymph osmolality 0.01 g/l Eades and Waring (2010)
Dreissena polymorpha 96 h Lipid peroxidation 1 g/l Quinn et al. (2011)
Mytilus edulis trossulus 8d Byssus strength 1 g/l Ericson et al. (2010)
Polystichum setiferum 48 h Mitochondrial activity 0.3 g/l Feito et al. (2012)
DNA quantication 0.03 g/l
Danio rerio embryos 90 min Lipid peroxidation 0.03 g/l Feito et al. (2012)

nr = not reported.
32 N. Vieno, M. Sillanp / Environment International 69 (2014) 2839
average of 175 g/g wet weight. Bioconcentration factors reported in
sh (rainbow trout) liver, kidney, gills and muscle tissue at aqueous
DCF concentration of 1 g/l have been reported as: 2732, 971, 763 and
69, respectively (Schwaiger et al., 2004). Kallio et al., 2010 studied the
occurrence of DCF and its metabolites in the bile of rainbow trout. In
aquarium conditions using spiked DCF of 1.9 g/l, DCF and its
metabolites (4-OH-DCF, 5-OH-DCF and glucuronides of DCF and the
metabolites) were detected in the bile samples (Kallio et al., 2010). Con-
centrations varied between 40 and 620 g/l. The total bioconcentration
factor (BFC) for DCF and its metabolites were estimated to be 950 (the
ratio between the total measured concentrations in the bile and the
measured exposure concentration in water). In the later study of the au-
thors (Brozinski et al., 2013), DCF could be detected in the biles of bream
and roach caught from a lake that receives municipal wastewater efu-
ents. DCF concentrations in the lake ranged from 22 to 302 ng/l whereas
the concentrations in the bile of bream and roach were up to 95 and
148 g/l, respectively, i.e. roughly 1000 times higher than the aqueous
concentrations.
In the environmental waters, DCF has been shown to rapidly
phototransform and to form several transformation products (Buser
et al., 1998; Svanfelt, 2013). Not only DCF itself but also these environ-
mental transformation products may pose a risk to aquatic organisms.
Schmitt-Jansen et al. (2007) reported a six-fold increase in phytotoxic-
ity evaluated using chlorophyte Scenedesmus vacuolatus when DCF
was exposed to sunlight for 53 h.
To summarize, humans excrete free and conjugated DCF and its me-
tabolites are excreted to municipal wastewater. DCF is shown to be
ubiquitous in municipal wastewater treatment plant efuents as well
as in the aquatic environment. DCF has been shown to pose harmful ef-
fects on organisms used in various test systems and to bioconcentrate in
sh and mussel at environmentally relevant concentrations. Therefore,
the load of DCF into the aquatic environment should be reduced.
2. Sorption of DCF to sewage sludge
In general, sorption of pharmaceuticals on sludge depends on the li-
pophilicity and acidity of the compound as well as the ambient condi-
tions such as pH, ionic strength, temperature and the presence of
complexing agents, and the properties of the sludge (Carballa et al.,
2005; Carballa et al., 2008). Sludge properties in a treatment plant
vary according to the type of plant and its method of operation. Primary
sludge is collected from the bottom of a primary sedimentation basin.
Secondary sludge is produced in the biological secondary treatment.
Its character and quantity depend on the biological treatment method
that can be for example activated sludge, MBR or moving bed biolm re-
actor (MBBR). Sludge removed from the treatment is often treated by
digestion (digested sludge) and/or composting (composted sludge).
Characteristics of sludge types differ greatly. For example, pH of the pri-
mary sludge is in general lower and fat and grease content higher than
that of activated or digested sludge. Compared to activated sludge, the
MBR sludge has a smaller particle size and thus a higher surface area
for adsorption (Kimura et al., 2007). Also, the content of inert matter
is often higher compared to activated sludge due to longer sludge age
applied in MBR process (Joss et al., 2006). All in all, sorption of com-
pounds is expected to be different to different sludge types.
Sorption of compounds to sludge occurs via absorption and adsorp-
tion. In absorption, the aliphatic and aromatic groups of the compound
interact hydrophobically with the lipophilic cell membrane of the
micro-organisms and the lipid fraction of the sludge (Ternes et al.,
2004a,b). In adsorption, the positively charged groups of the chemical
interact electrostatically with the negatively charged surfaces of the
micro-organisms (Ternes et al., 2004a,b). Sorption accounts both ab-
sorption and adsorption and it can be estimated by Kd value. It is the
ratio of compound's concentration in the solid and in the aqueous
phase at equilibrium conditions (Carballa et al., 2005). According to
Ternes et al. (2004a), sorption of a compound to sludge can be
considered negligible when Kd value is b 500 l/kgSS (i.e. logKd b 2.7). Ac-
cording to Joss et al. (2005), Kd should be over 300 l/kg (i.e. logKd N 2.5)
for an efcient sorption.
Diclofenac is slightly soluble in water and has a moderately low
octanolwater coefcient (Table 1). Measured and calculated logKd
values of DCF to different sludge types in wastewater treatment vary be-
tween 1.2 and 2.7 (Table 1) (Carballa et al., 2008; Radjenovic et al.,
2009; Ternes et al., 2004a,b). Highest Kd values have been reported for
primary sludge. In Ternes et al., 2004a, sorption of diclofenac was b5%
to secondary sludge whereas the sorption was 515% to primary sludge.
Radjenovic et al. (2009) also measured higher sorption potential of DCF
to primary than to secondary sludge. In a sewage treatment plant in
Sweden, DCF concentration was noted to decrease by 50% during the
pre-treatment of sewage by grit removal and primary sedimentation
(Zorita et al., 2009). This behavior suggests that DCF primarily interacts
with the sludge via adsorption where pH plays a signicant role (Ternes
et al., 2004a). The carboxylic acid moiety of DCF is negatively ionized at
neutral pH and hence the compound repels the negatively charged
sludge. At acidic pH, DCF becomes electronically neutral thus allowing
it to adsorb to the sludge. In primary treatment, process pH is in general
lower than in biological treatment unit. For example, in Ternes et al.
(2004a), pH values were 6.6 and 7.5 at the primary and secondary treat-
ment, respectively. Urase et al. (2005) noted that in MBR reactor the
lower pH enhanced the DCF removal. Removal was only 10% at a pH
range of 6.88.0 but was increased to 80% at the MBR reactor with pH
in the range of 4.35. At neutral pH the carboxyl group of DCF is disso-
ciated and consequently the compound is negatively charged. The au-
thors suggested that the removal was due to adsorption to sludge and
the adsorption was promoted by the lower pH. Additionally, Urase
and Kikuta (2005) noted in their laboratory batch experiments that
partitioning of DCF to activated sludge increased by more than 20
times when pH was decreased from 6.7 to 4.4.
Sorption of DCF to MBR sludge has been found to be slightly higher
than to activated sludge (Table 1). However, there are differences be-
tween MBR sludges. Radjenovic et al. (2009) measured lowest concen-
tration of DCF in the sludge that was collected from an MBR operating at
prolonged SRT and high concentration of suspended solids. The authors
suggested that this was due to the higher biodegradation potential of
the sludge resulting in lower amount of soluble DCF for sorption.
At wastewater treatment plants, sorption of DCF to sludge have been
reported but to a low extent (Gracia-Lor et al., 2012; Kimura et al., 2007;
Martn et al., 2012; Surez et al., 2012; Verlicchi et al., 2012). Samaras
et al. (2013) reported that 819% of DCF was bound to particles in raw
wastewater. In dewatered sludge, DCF concentration was 30 g/kg
and in raw and digested sludge below the detection limit. Yu et al.
(2013) reported a mean concentration of DCF in sewage sludge as
48.4 g/kg. Martn et al. (2012) found that DCF concentrations were al-
ways below the detection limits (1.2233.1 g/kg) in different sludges
collected from wastewater treatment plant. In activated sludge, concen-
trations of 150450 g/kg have been reported (Radjenovic et al., 2009;
Ternes et al., 2005). In primary sludge, Radjenovic et al. (2009) mea-
sured DCF at a concentration of 200 g/kg and at MBR sludge at
100175 g/kg. In digested sludge, concentrations of 200 g/kg have
been reported (Carballa et al., 2007; Ternes et al., 2005). Radjenovic
et al. (2009) calculated that DCF load in the aqueous efuent and in
the treated sludge from a Spanish wastewater treatment plant was in
average of 46.5 g/d and 6.4 g/d, respectively. Thus, only about 10% of
the environmental load from wastewater treatment plant can be esti-
mated to occur via solids.
2.1. Removal of DCF in sludge treatment
Excess sludge from the treatment process is often anaerobically
digested to stabilize sludge and to produce methane for energy produc-
tion. Depending on the temperature, digestion can be mesophilic (opti-
mum T = 2540 C) or thermophilic (optimum T = 5565 C). Higher
 N. Vieno, M. Sillanp / Environment International 69 (2014) 2839
temperature accelerates biochemical reactions and has been noted to
slightly enhance the removal of solids and organic material in compar-
ison to mesophilic process (Carballa et al., 2007). According to
Carballa et al. (2007), DCF removal rates in both mesophilic and ther-
mophilic processes are similar and can reach 80%. According to
Samaras et al. (2013), other NSAID ibuprofen and naproxen were re-
moved by N80% during mesophilic sludge digestion. However, DCF con-
centration was already below the detection limit in the raw sludge and
thus process performance could not be evaluated. However, in Carballa
et al. (2007) high removal rates were only detected at long operating
times suggesting that sludge acclimation followed by broader biodiver-
sity is needed for DCF removal. Sorption of DCF to digested sludge has
been found to be fairly low (Table 1) even though Carballa et al.
(2008) had the following hypothesis. The digested sludge has a different
composition, structure and morphology and it can be modied by dif-
ferent pretreatment techniques. Thus, the adsorption of DCF to digested
sludge could be higher than to primary and secondary sludge. Therefore,
primary elimination method in sludge treatment is most likely biotrans-
formation rather than sorption.
3. Biological elimination and transformation of DCF
The objectives of the biological treatment of municipal wastewater
are to coagulate and remove the non-settleable colloidal solids, to re-
duce the organic content and the nutrients nitrogen and phosphorous.
These are accomplished using a variety of micro-organisms, principally
bacteria, of which there may be 300 species present. The predominant
group of bacteria is heterotrophs that mainly feed on organic carbon
molecules. Inorganic matter is taken in by autotrophs, such as ammonia
oxidizing bacteria that oxidize ammonia into nitrite. Heterotrophs often
out-compete autotrophs which have lower growth rate and are often
more sensitive to process conditions and variations.
Nitriers are the most important group of autotrophic bacteria in bi-
ological wastewater treatment. Several bacteria genera are responsible
for nitrication, for example Nitrosomonas, Nitrobacter and Nitrospira.
Nitrosomonas oxidizes ammonia to nitrite (NO 2 ), which is further con-
verted to nitrate (NO 3 ) by Nitrobacter and Nitrospira. Nitrifying bacteria
are sensitive organisms whose optimum pH and temperature ranges
are narrow. They are also sensitive to inhibitors. In fact, some pharma-
ceuticals have been noted to inhibit the performance of ammonia oxi-
dizing bacteria (Wang and Gunsch, 2011). For example, gembrozil
and naproxen were noted to decrease ammonia removal by 45% in a
pilot scale sequencing batch reactors mimicking wastewater treatment
plants operations (Wang and Gunsch, 2011). COD removal was not af-
fected by the addition of pharmaceuticals, suggesting that heterotrophic
bacteria are more robust to these compounds. Kraigher et al. (2008) no-
ticed that addition of pharmaceutical mix of ibuprofen, naproxen,
ketoprofen, diclofenac and clobric acid into a pilot scale wastewater
treatment system reduced the diversity in bacterial communities al-
ready at an individual pharmaceutical concentration of 5 g/l. Most sig-
nicantly, genus Nitrospira was found only in the reactor without
pharmaceuticals representing 8% of the total community. Genus
Nitrospira plays a key role in nitrite oxidation during biological waste-
water treatment. Reduction in species diversity is often reported in re-
sponse to stress factors, e.g. phenol shocks and elevated temperatures.
A reduction in bacterial diversity may affect the essential functions of
the activated sludge wastewater treatment systems. However, in the
later study of the authors (Kraigher and Mandic-Mulec, 2011) addition
of pharmaceuticals in the reactors was not noted to decrease the nitri-
cation activity of activated sludge bacteria and the removal of ammonia
was constantly N 90%. However, in reactors with pharmaceutical con-
centration of 50 g/l, concentrations of N(NO
2 + NO3 ) was signi-
cantly higher than in reactors with no pharmaceuticals and in reactors
with higher pharmaceutical concentration (i.e. 200 and 500 g/l). The
authors could relate the high concentration of N(NO
2 + NO3 ) into
the abundance of Nitrospira sublineage II in reactors with 50 g/l of
33
pharmaceuticals. Other reactors only contained Nitrospira sublineage I.
They suggested that, at lower concentrations, Nitrospira sublineage II
could use pharmaceuticals as carbon source or could co-metabolize
them. As the concentrations increase, sublineage I or some other com-
petitor could co-metabolize pharmaceuticals and the advantage of
Nitrospira II sublineage is lost. Other explanation that was given by
the authors was the suggestion that pharmaceuticals could negatively
inuence the activity and/or structure of denitrication bacteria at low
pharmaceutical concentration. This could have caused the increase in
nitrate concentration and changes in Nitrospira community structure.
However, no nitrite accumulation and thus inhibition of nitrite oxida-
tion by pharmaceuticals was noted. The authors did not consider the
possibility of abiotic transformation of pharmaceuticals and, more spe-
cically, the potential of some pharmaceuticals, such as diclofenac, to
undergo abiotic nitration processes in the presence of nitrite. This phe-
nomenon is further discussed in Section 3.2. At higher pharmaceutical
concentration the consumed nitrite into abiotic processes is also
increased.
The biological elimination of pharmaceuticals in municipal waste-
water treatment plants could occur by direct metabolization or by co-
metabolization. In the former, bacteria use the compound as their
primary carbon source whereas in the latter, bacteria break down or
partially convert the compound but do not use it as the primary carbon
source (Jones et al., 2007; Ternes et al., 2004a,b). Co-metabolism occurs
via enzymes that bacteria secrete to break down large organic mole-
cules into monomers that are small enough to be ingested. Many of
these enzymes have the potential to degrade or to transform small or-
ganic pollutant molecules present in wastewater. For example, E. coli
secrete -glucuronidase enzyme that is capable to deconjugate the -
glucuronated pharmaceuticals excreted by the human body. Thus, this
could result in releasing the active pharmaceutical into the wastewater.
Ammonia monooxygenase (AMO) is secreted by autotrophic ammonia
oxidizing bacteria during the oxidation of ammonium to hydroxylamine
in aerobic nitrication. Hydroxylamine is further converted to nitrite.
AMO is capable of degrading pollutants such as pharmaceuticals via hy-
droxylation. It has been suggested to be one of the main enzymes in co-
metabolic reactions for removal of micropollutants from wastewater
(Fernandez-Fontaina et al., 2012; Roh et al., 2009; Tran et al., 2009).
The ability of bacteria to secrete a particular enzyme may be latent, i.e.
the bacterium requires the presence of the particular compound in the
water to switch on the genes for the synthesis of the enzyme required
for its digestion. In case of process changes of biological wastewater
treatment such as increase in sludge age or sudden increase in toxic
compounds, those species of bacteria that have the ability to secrete
the enzymes to break down a novel food source will grow more rapidly.
This process is known as adaptation or acclimation.
Current assumption is that due to their trace level concentrations in
municipal wastewater, pharmaceuticals are degraded by microbial en-
zymes through co-metabolism (Fernandez-Fontaina et al., 2012;
Onesios et al., 2009; Quintana et al., 2005; Roh et al., 2009; Tran et al.,
2009). For example, Quintana et al. (2005) carried out metabolic and
co-metabolic biodegradation tests for pharmaceuticals according to
ISO 7827 using sludge, which was drawn from a reactor treating waste-
water in which all the studied pharmaceuticals were found. They con-
cluded that when pharmaceuticals were used as the sole substrate no
degradation occurred during the 28-d test period. On the other hand,
in the co-metabolism test, bezabrate, naproxen and ibuprofen were
degraded by 3096% in 28 days.
3.1. Biotransformation mechanism of DCF
Generally, biodegradation of DCF has been found to be slow or non-
existing in biodegradability studies (Buser et al., 1998; Joss et al., 2005;
Lee et al., 2012; Perez and Barcelo, 2008; Quintana et al., 2005).
Quintana et al. (2005) concluded that DCF could not be degraded
neither through metabolic nor co-metabolic route. Also, low
34 N. Vieno, M. Sillanp / Environment International 69 (2014) 2839

Table 4 Tran et al. (2009), increase in initial ammonia concentration was no-
Values of biological degradation constant kbiol of diclofenac at different biological process ticed to signicantly increase the DCF removal. Since increase in ammo-
congurations.
nia concentration increases the AMO concentration the increase could
kbiol (l g1SS d1) Biological process conguration Reference be suggested to be due to degradation by AMO. In addition, inhibition
b0.1 Conventional activated sludge Joss et al. (2006) of AMO by allylthiourea addition was noted to decrease the DCF remov-
b0.1 Membrane bioreactor Joss et al. (2006) al from about 75% to 25%. On the other hand, the authors performed a
b0.1 Anoxic + aerobic Surez et al. (2012) test where they added sodium acetate into the reactor to induce the
0.04 Anoxic conditions Surez et al. (2010)
growth of heterotrophic bacteria. DCF removal was signicantly in-
1.2 Nitrifying conditions Surez et al. (2010)
0.310.52 Nitrication culture Tran et al. (2009) creased after the acetate addition suggesting the signicant role of het-
erotrophs in DCF biodegradation. It therefore seems, that both
autotrophic and heterotrophic bacteria contribute to the biological deg-
radation/transformation of DCF. Further research is needed to identify
biodegradation of the main human metabolite 4-OH-DCF has been re- the micro-organism communities that are able to degrade DCF. This in-
ported (Lee et al., 2012). Its pseudo-rst order rate constant under biotic formation could be used in bioaugmentation of wastewater with these
conditions has been measured as 0.018 d1 which translates into a half- communities to enhance the DCF biotransformation. Rodrguez-
life of nearly 40 d (Lee et al., 2012). Biological degradation constant, Rodrguez et al. (2012) used bioaugmentation of sewage sludge with
kbiol, can be used to estimate the biodegradation potential of a com- fungi Trametes versicolor to degrade several pharmaceuticals. During
pound. Measured kbiol values for DCF are presented in Table 4. Joss the 42 d bioremediation experiment, DCF was eliminated by 22% with-
et al. (2006) suggested a classication scheme for micropollutants out the addition of T. versicolor. Elimination was increased to up 61% by
based on the kbiol (as l g1SS d1): using the fungal treatment.

kbiol b 0.1: no substantial biodegradation (b20%) 3.2. Biotic and abiotic transformation products
0.1 b kbiol b 10: partial biodegradation (2090%)
kbiol N 10: ready biodegradation (N90%). It seems that the degradation pathway of DCF in biological wastewa-
ter treatment is a complex process. Unfortunately only few of the bio-
For DCF, Joss et al. (2006) as well as Surez et al. (2012) measured degradation studies have reported on the structural identity of DCF
kbiol values of b 0.1 meaning that the compound does not undergo bio- metabolites and other transformation products (Table 5). Two of the
degradation. Surez et al., 2010 applied following classication scheme
to estimate biological degradation of pharmaceuticals:
Table 5
Metabolites of biological transformation of diclofenac and their suggested molecular
kbiol b 0.5: hardly biodegradable structures. Figure after the letter M refers to the molecular weight of the metabolite.
0.5 b kbiol b 1: moderately biodegradable
Molecular structure or chemical formula Information about the metabolite
1 b kbiol b 5: highly biodegradable
kbiol N 5: very highly biodegradable. Metabolite M190
Biological wastewater treatment
Perez and Barcelo (2008)
According to Surez et al. (2010) kbiol of DCF was 0.04 l g1SS d1, i.e.
no biodegradation, in anoxic conditions. However, kbiol value of 1.2 l g
1 1
SS d was measured for DCF in aerobic nitrifying conditions suggest-
ing high biodegradation potential of DCF. Also, Tran et al. (2009) noted Metabolite M250
that DCF is moderately biodegradable in experiments that used Laboratory-scale biological treatment
with activated sludge
enriched nitrifying activated sludge (kbiol of 0.310.52 l g 1SS d 1).
Isomeric structure, the position and
Surez et al. (2010) concluded that enrichment of specic nitrifying nature of CH2-group not dened.
bacteria in the reactor were probably responsible of the high biodegra- Kosjek et al. (2009)
dation potential of DCF.
Good nitrifying activities have been noted to increase the biodegra-
C14H11NO2Cl2 Metabolite M275
dation rates of several micropollutants (Fernandez-Fontaina et al.,
Laboratory-scale biological treatment
2012). It has been suggested that the increased removal of several phar- with activated sludge
maceuticals under nitrifying conditions could be due to their degrada- Kosjek et al. (2009)
tion by ammonia monooxygenase secreted by ammonia oxidizing Metabolite M278
bacteria (Fernandez-Fontaina et al., 2012; Tran et al., 2009). Although, Laboratory-scale biological treatment
with activated sludge
ibuprofen which is a highly biodegradable anti-inammatory pharma- Kosjek et al. (2009)
ceutical, seems to be degraded primarily by heterotrophic bacteria
(Roh et al., 2009, Tran et al., 2009). In the case of DCF, some studies
Metabolite M324
argue that nitriers are not the sole organisms responsible for DCF bio-
Biological wastewater treatment
degradation (De Graaff et al., 2011; Fals et al., 2012). Fals et al. (2012) Perez and Barcelo (2008)
noted low or negligible removal of pharmaceuticals in the reactor,
which contained carriers from a partial nitritation/anammox sludge li-
quor treatment. According to the authors, this indicates that the ammo-
nia oxidizing bacteria have very limited ability to degrade/transform
DCF even at high ammonia concentration and realistic nitritation Metabolite M340
Biological wastewater treatment
rates. They concluded that nitrifying bacteria contribute very little or
Perez and Barcelo (2008)
not at all to the removal of DCF in nitrifying biological treatment sys-
tems. Consistent with this study are the results reported by De Graaff
et al. (2011) who studied the fate of hormones and pharmaceuticals in
anaerobic treatment, partial nitritation reactor and anammox reactor.
No signicant removal of DCF was detected during the treatment. In
 N. Vieno, M. Sillanp / Environment International 69 (2014) 2839
metabolites, i.e. M340 and M324 can be related to nitrifying wastewater
bacteria. Metabolite M324 is formed by O-nitritation of the hydroxyl
group of the carboxylic acid moiety in DCF. According to the authors
(Perez and Barcelo, 2008), this is an untypical metabolic pathway
brought about by micro-organisms. Metabolite M340, on the other
hand, was concluded to be a product of microbial nitration at one of
the aromatic rings. In general, metabolites undergoing ring hydroxyl-
ation has not yet been reported for DCF which would have been an ev-
idence on monooxygenase-mediated reaction.
There may be another explanation for the formation of nitro-
products during wastewater treatment. Namely, these transformation
products of DCF during the nitricationdenitrication process could
form via abiotic nitration in the presence of nitrite rather than via bio-
logical degradation. This is supported by the study of Gaulke et al.
(2008) who conducted batch tests on the biodegradation of 17-
ethinylestradiol (EE2) with pure cultures of ammonia oxidizing bacteria
from Nitrosomonas and Nitrosospira. They reported that co-metabolic
degradation of EE2 occurred neither with the addition of ammonia ni-
trogen at 10 mg/l nor without the addition. On the other hand, ammonia
was completely oxidized to nitrite. EE2 could be degraded to some ex-
tent when ammonia concentration was increased to 200500 mg/l.
The authors concluded that EE2 transformation was not due to the en-
zymatic attack by the bacteria but rather by abiotic nitration of EE2
with nitrite produced by the bacteria. Similar could happen to DCF in
the wastewater nitrication unit. Also, it has been suggested that abiotic
nitration products could return to parent compound when the concen-
tration of nitrite reduces (Barbiere et al., 2012). Barbiere et al. (2012)
studied the effect of subsurface articial groundwater inltration
under nitrate reducing conditions on pharmaceuticals. A sudden drop
after the 1.5 d of incubation to about 50% of initial concentration of
DCF was noted. However, by day 10, the concentration of DCF had
returned to the initial concentration. Thus, after 10-day incubation, no
overall removal of DCF was noted. The authors also observed that this
drop in concentration occurred currently and oppositely to the reduc-
tion of nitrite. Similar trend was noted to sulfamethoxazole, which is
also an aromatic amine substance. Authors could identify a degradation
by-product to be a nitro analog of DCF, which was suggested to occur via
nitration at one of the aromatic rings and thus have the structure of the
metabolite M340 (see Table 5). The authors concluded, that a phenom-
enon that has been previously noted to other aromatic amines, namely
formation of nitroproducts in the presence of nitrite, occurs to DCF.
Nitro-derivatives return to parent compounds when the concentration
of nitrite drops. This phenomenon could also occur in wastewater treat-
ment during nitrication and denitrication process. This can lead to
wrong estimation of the actual treatment efciency of DCF as nitro
100%
90%
80%
70%
60%
50%
40%
36%
30%
20%
10%
0%
Conventional activated sludge
Fig. 2. Elimination rates in wastewater treatment plants with different biological treatment processes. Filled symbols and the percentage value refer to the average elimination rates for the
treatment method in question. Note that this percentage does not contain the negative elimination rates reported in Clara et al. (2005a), Kosma et al. (2014) and Zorita et al. (2009). Ref-
erences are listed in Table S1.
35
derivatives can transform back to the parent compounds when released
into the environment.
4. Elimination of DCF during biological wastewater treatment
Generally, high variation in elimination rates has been noted in mu-
nicipal wastewater treatment plants (Table S1 and Fig. 2). Considering
the complex picture of DCF biodegradation/biotransformation
discussed in the previous section it seems only natural that high varia-
tion occurs when only the inuent and efuent concentrations are con-
sidered in the calculations of elimination rates. Elimination rates of up to
about 80% can be reached at the treatment; however, values in the
range of 2050% are more common. Also, increase in DCF concentration
and thus negative elimination has been reported (Clara et al., 2005a;
Zorita et al., 2009). Zorita et al. (2009) measured an increase in DCF con-
centration from 100 to 485 ng/l in activated sludge treatment consisting
of subsequent anoxic and aerobic process units for biological nitrogen
removal. The authors suggested that this was due to deconjugation of
glucuronide or sulfate conjugates of DCF and/or desorption of DCF
from the particles. According to Lee et al. (2012) a rapid deconjugation
of the glucuronide conjugate to release DCF can occur at wastewater
treatment. In microbiological degradation tests, they noted that the
glucuronide-DCF deconjugated to form equimolar DCF within 7 d of in-
cubation. Reaction rate constant were 0.28 d1 (t1/2 2.5 d) in the in-
oculum and 0.12 d1 (t1/2 5.8 d) under sterile conditions. This
suggests that abiotic hydrolysis occurs but the reaction is enhanced by
micro-organisms.
According to Stierlin and Faigle (1979), b 1% of the administered DCF
dose is excreted as free DCF and about 11% as conjugated DCF (glucuro-
nide and sulfate). If deconjugation occurs, a 10-fold increase in DCF con-
centration is possible in the sewer and/or in the biological wastewater
treatment. One possible source of DCF could also be biological transfor-
mation of another analgesics, namely aceclofenac. Perez and Barcelo
(2008) noted that this compound underwent a rapid ester cleavage to
DCF during biological wastewater treatment. However, aceclofenac is
not as common a pharmaceutical as DCF. For example, its consumption
has not been reported in Finland or in Norway (Finnish Medicines
Agency, 2013; Sakshaug, 2012). Aceclofenac is sold under the commer-
cial names of Biofenac, Airtal, Barcan, Cartrex, Falcol, Gerbin, Gladio,
Kafenac, Preservex, Sanein and Sovipan. Concentrations measured
in the wastewater have also been signicantly lower than DCF
concentrations. In a Spanish wastewater treatment plant, aceclofenac
concentrations in average of 33 ng/l were measured whereas DCF con-
centrations were in average of 349 ng/l (Perez and Barcelo, 2008).
48%
36%
Activated sludge with biological nutrient removal Membrane bioreactor
36 N. Vieno, M. Sillanp / Environment International 69 (2014) 2839
Therefore, aceclofenac may not be the main source for increased DCF
concentrations reported in wastewater treatment.
4.1. Effect of process conguration
Nowadays, conventional activated sludge (CAS) and activated
sludge with biological nutrient removal (BNR) are the most commonly
applied biological process in full-scale wastewater treatment plants.
CAS is an aerobic suspended-growth treatment process. BNR is s
suspended-growth treatment process that combines aerobic, anaerobic
and anoxic treatment units. Recently, interest in membrane bioreactors
(MBRs) has increased. MBR is a membrane process using micro- or
ultraltration together with suspended-growth biomass. The most im-
portant advantage of MBR over CAS or BNR is the complete retention
of suspended solids and thus low efuent turbidity. In addition, MBR
has less sludge production, 5070% smaller footprint size and better ef-
uent quality in terms of bacteria, viruses and sometimes also DOC and
COD (Bernhard et al., 2006; De Wever et al., 2007). Also, MBR can be
used together with powdered activated carbon (PAC), which reduces
the membrane cleaning or replacement frequency and could enhance
the removal of micropollutants as well (Nguyen et al., 2013). For exam-
ple, in Nguyen et al. (2012), MBR alone was noted to eliminate DCF by
426%. The addition of PAC increased the removal to 96%. Disadvantages
of MBR over CAS and BNR are higher cost, higher requirements in oper-
ation and maintenance as well as power consumption compared to con-
ventional system (Clara et al., 2005b). A moving bed biolm reactor
(MBBR) is an example of attached-growth biomass process where bio-
mass grows on specially designed carriers that move freely with the re-
actor water volume and biolm is grown on the carrier surfaces.
Advantages of MBBR are simplicity, compactness, growth of aerobic
and anaerobic organisms in the same system, and negligible hydraulic
headloss (Zupanc et al., 2013).
Table S1 and Fig. 2 show that no single biological treatment congu-
ration outperforms the other. For MBBR, only one study was found that
reported the removal of DCF (Zupanc et al., 2013). In the study, MBBR
resulted in 74 to 85% removal of DCF compared to 36% in activated
sludge system. The authors suggested that the micro-organisms in the
biolm that was developed on the surfaces on carriers were able to ex-
ploit DCF as organic substrate.
According to Fig. 2, similar average removal rates calculated from lit-
erature studies were reported for CAS and BNR processes. However,
many individual studies report a higher removal of DCF under nitrifying
conditions (Maeng et al., 2013; Surez et al., 2010; Tran et al., 2009). On
the other hand, DCF is not degraded under anoxic conditions, i.e. under
denitrifying conditions (Surez et al., 2010). Since only inuent and ef-
uent concentrations have been used to evaluate the elimination rates,
the formation of by-products as well as the deconjugation and subse-
quent release of DCF have not been considered. As was discussed in
the previous section, the abiotic nitration of DCF occurs in the presence
of nitrite (NO
2 ). During CAS, nitrication is often incomplete, i.e. nitrite
does not fully oxidize to nitrate. Thus, nitrite is free to react with DCF
and could result in reduction of monitored DCF concentration. It should
be noted, that nitro-DCF can degrade and release DCF in the following
treatment units or in the environment. During nitrication, nitrite is
rapidly further oxidized to nitrate (NO 3 ), which does not react with
DCF and form nitro-DCF. Thus, more DCF could therefore be released
from the reactor compared to the CAS reactor.
Some studies have reported higher elimination of DCF in MBR pro-
cesses compared to CAS, whereas others have noted no difference be-
tween the treatments. Radjenovic et al. (2009) reported a higher
elimination of DCF in MBR process (in average 6366%) over the CAS
process (in average of 22%). Kimura et al. (2007) noted a removal of
40% in CAS and up to 80% in MBR. Bernhard et al. (2006) compared
the optimized laboratory scale MBR and CAS treatment. They reported
slightly better elimination (in average of 58%) in MBR than in CAS (in
average of 24%) process. On the other hand, Clara et al. (2005b)
concluded that there are no correlation between the treatment method
and DCF elimination and that MBR and CAS both showed low removal of
DCF. Similar results were obtained by Joss et al. (2005) who reported
low (i.e. 2040%) removal of DCF in CAS, MBR and xed bed reactors.
Also Xue et al. (2010) recorded low DCF elimination rates with all tested
treatment congurations (anaerobic, anoxic, arobic and membrane).
Further, Kimura et al. (2005) reported a poor removal of DCF in both
CAS and two different types of MBRs, namely hybrid MBR and conven-
tional MBR.
The studies that report better performance of MBR over CAS or BNR,
suggest that higher biomass content and the longer SRTs applied in MBR
could be the reason for its better performance (Kimura et al., 2007; Sui
et al., 2011). Higher biomass leads to a lower food to micro-organisms
ratio and the relative shortage of biodegradable organic matter may
force micro-organisms to metabolize more recalcitrant compounds in
the sewage (Sui et al., 2011). Longer SRTs allow bacterial population
to become more diversied and more capable of degrading DCF either
by direct metabolism or by co-metabolic degradation via enzymatic re-
actions. Also, MBR sludge has a smaller particle size than activated
sludge and thus a higher surface area for adsorption. Even though ad-
sorption of DCF to sludge is low, Kimura et al. (2007) suggested that
the higher microbial activity in MBR sludge enhances the biodegrada-
tion of the adsorbed DCF.
A signicant advantage of MBR over CAS or BNR is that micro-
pollutant removal in general has been noted to be less sensitive to oper-
ational variables (De Wever et al., 2007). According to De Wever et al.
(2007), if the micropollutant feed is stopped for some period and feed
started again, MBR resumes much faster than CAS. This is probably
due to the retention of bacteria required to degrade micropollutants
by the MBR membrane. In CAS, the washout of these bacteria is faster
and more pronounced. MBR can therefore respond more quickly to tem-
poral uctuations in micropollutant inuent concentrations.
4.2. Effect of process parameters
4.2.1. Solids retention time (SRT)
Solids retention time (also known as sludge retention time or sludge
age) represents the mean residence time of micro-organisms in a reac-
tor. Only organisms that are able to reproduce themselves during this
time can be detained and enriched in the system. High SRT allow the en-
richment of slowly growing autotrophic bacteria such as nitriers.
Therefore, in BNR reactors SRT can exceed 20 days whereas in CAS
processes, SRTs normally range between 5 and 15 days. It has been
suggested that the presence of more diversied bacterial populations
induced by longer SRTs would enhance the degradation of micro-
pollutants either by direct metabolism or by co-metabolic degradation
via enzymatic reactions. For example, it has been reported that an in-
crease in SRT would increase the elimination of some pharmaceuticals,
such as gembrozil, ketoprofen, clobric acid and EE2 (Clara et al.,
2005a; Kimura et al., 2007; Maeng et al., 2013; Surez et al., 2010,
2012). For DCF, the effect of SRT is not that straightforward. Joss et al.
(2005) reported no enhancement of elimination rates of DCF even
when extreme SRTs (N 60 d) were applied. Similarly, Surez et al.
(2012) and Clara et al. (2005b) noted no correlation between the elim-
ination of DCF and SRT. Reif et al. (2008) applied an SRT of 72 d in MBR
treatment and noted no elimination of DCF. Majority of the studies
reporting higher elimination of DCF at higher SRTs can be related to
MBR processes. Bernhard et al. (2006) noted an increase in DCF elimina-
tion with increasing SRT using MBR reactor. DCF removal was 838%
when SRT was 2048 d, 59% at SRT of 62 d and 53% at SRT of 322 d.
Kimura et al. (2007) studied the removal DCF in real WWTP applying
activated sludge (AS) and in membrane bioreactor (MBR). Sludge ages
were 7 d for the AS and 15 d and 65 d, respectively, for the two tested
MBRs. The authors noted that the removal of all the pharmaceuticals
were the highest at the MBR with the sludge age of 65 d. DCF was re-
moved by 40% at AS and MBR (15 d) but up to 80% at MBR (65 d). The
 N. Vieno, M. Sillanp / Environment International 69 (2014) 2839
only exception to this pattern was presented in Fernandez-Fontaina
et al. (2012), where increasing SRT to N 150 d has resulted in 70% elim-
ination of DCF in laboratory scale AS process working under nitrifying
conditions.
Generally, it seems that increase in SRT may not be a feasible method
to increase the elimination of DCF under normal WWTPs. It has been
noted that only extremely high SRTs N 150 d may be needed to obtain ef-
cient DCF elimination in activated sludge plants (Fernandez-Fontaina
et al., 2012). Application of this high SRT values in real life CAS or BNR
plant is unrealistic.
4.2.2. Hydraulic retention time (HRT)
Hydraulic retention time is the residence time of the aqueous sew-
age in a reactor or in the entire process. Increase in HRT has been report-
ed to increase the removal of certain pharmaceuticals, such as
ibuprofen, ketoprofen, atenolol, sotalol, metoprolol, uoxetine and
roxithromycin probably due to increased contact time between the
micro-organisms and the water to be treated (Fernandez-Fontaina
et al., 2012; Maurer et al., 2007; Tauxe-Wuersch et al., 2005). Gros
et al. (2010) concluded that pharmaceuticals that are biodegradable
(high kbiol, i.e. low half-life t1/2) but have a low tendency to sorb to
sludge, are the most inuenced by the changes in HRT. Those
micropollutants that have a half-life of less than the HRT can be ef-
ciently removed in the biological reactor (Verlicchi et al., 2012; Gros
et al., 2010). Surez et al. (2012) estimated that a half-life of DCF for
WWTPs working with biomass concentration in the range of 24 g/l
would be 23.5 days. According to them, only those plants operating
at high enough HRT are expected to be able to degrade it to a certain ex-
tent. In their study, the applied HRT was only 1 d and the authors sug-
gested that the poor removal of DCF was due to this. HRTs in full scale
WWTPs are normally measured in hours not in days. Therefore, increase
in HRT could signicantly enhance the elimination of DCF. In fact, in
Clara et al. (2005a), 70% removal of DCF at a plant with HRT of 13 d
was measured, whereas negligible removal was measured at a plant op-
erating at HRT b 1.2 d.
5. Conclusions
Elimination of DCF has been shown to vary greatly in laboratory,
pilot and full-scale wastewater treatment processes. To some extent,
the compound is shown to adsorb to primary sludge but generally bio-
transformation is considered as the main elimination mechanism in
wastewater treatment. However, since DCF is only moderately or poorly
biodegradable incomplete elimination during the conventional waste-
water treatment applying CAS or BNR processes can be expected. Elim-
ination could be enhanced by the following means:
Application of membrane bioreactor processes or attached-growth
biomass process may result in higher DCF elimination. There are
also other advantages that favor the use of these processes over
the conventional suspended-growth biomass processes.
Increase of HRT to N 23 days could enhance the elimination of DCF
since this would increase the contact time of water with the bio-
mass.
Enrichment of DCF degrading micro-organisms into the bioreactor.
This could be done by applying SRTs N150 d which may not be real-
istic at full-scale wastewater treatment plants. Also, bioaugmenta-
tion could be used, i.e. addition of cultured micro-organisms able
to degrade DCF into the biological process.
However, due to the deconjugation processes and formation of sev-
eral biotransformation products as well as abiotic nitro-products, degra-
dation pathways need to be further investigated to fully understand the
fate of DCF in biological wastewater treatment. Further research is also
needed to identify the microbial communities degrading DCF that
could be used in bioaugmentation.
37
In the future, DCF may be classied as priority substance with EQS
values ranging from 10 to 100 ng/l. These values were proposed during
the revision of EU's priority substance directive during 20122013.
Many studies reviewed here reported DCF concentrations of
N1000 ng/l in WWTP efuents. Thus, especially in the rivers that are
highly inuenced by WWTP efuents, DCF concentrations can exceed
100 ng/l. To summarize, low enough concentrations of DCF in the
WWTP efuents may not be possible to obtain by optimizing the
existing biological processes only. To enhance DCF elimination, we
need to further investigate and develop effective and cost-efcient ter-
tiary treatment methods, such as methods based on oxidation or
adsorption.
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.envint.2014.03.021.
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