Journal of Hazardous Materials 239240 (2012) 4047

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Journal of Hazardous Materials

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Occurrence of pharmaceutical compounds in wastewater and sludge from

wastewater treatment plants: Removal and ecotoxicological impact of
wastewater discharges and sludge disposal

J. Martn, D. Camacho-Munoz, J.L. Santos, I. Aparicio , E. Alonso
Department of Analytical Chemistry, Escuela Politcnica Superior, University of Seville, C/Virgen de frica 7, E-41011 Seville, Spain

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

Article history: The occurrence of sixteen pharmaceutically active compounds in inuent and efuent wastewater and
Received 11 January 2012 in primary, secondary and digested sludge in one-year period has been evaluated. Solidwater parti-
Received in revised form 25 April 2012 tion coefcients (Kd ) were calculated to evaluate the efciency of removal of these compounds from
Accepted 29 April 2012
wastewater by sorption onto sludge. The ecotoxicological risk to aquatic and terrestrial ecosystems, due
Available online 7 May 2012
to wastewater discharges to the receiving streams and to the application of digested sludge as fertil-
izer onto soils, was also evaluated. Twelve of the pharmaceuticals were detected in wastewater at mean
Keywords:
concentrations from 0.1 to 32 g/L. All the compounds found in wastewater were also found in sewage
Pharmaceutically active compound
Wastewater
sludge, except diclofenac, at mean concentrations from 8.1 to 2206 g/kg dm. Ibuprofen, salicylic acid,
Sludge gembrozil and caffeine were the compounds at the highest concentrations. Log Kd values were between
Solidwater partition coefcient 1.17 (naproxen) and 3.48 (carbamazepine). The highest ecotoxicological risk in efuent wastewater and
Environmental risk assessment digested sludge is due to ibuprofen (risk quotient (RQ): 3.2 and 4.4, respectively), 17-ethinylestradiol
(RQ: 12 and 22, respectively) and 17-estradiol (RQ: 12 and 359, respectively). Ecotoxicological risk after
wastewater discharge and sludge disposal is limited to the presence of 17-estradiol in digested-sludge
amended soil (RQ: 2.7).
2012 Elsevier B.V. All rights reserved.

1. Introduction on the characteristics of sewage sludge. The inuence of sorption

Pharmaceutically active compounds have become environment
pollutants of emerging concern because their intrinsic biolog-
ical activity which may cause adverse effects to aquatic and
terrestrial ecosystems, particularly at chronic exposure. Pharma-
ceutical compounds reach wastewater treatment plants (WWTPs)
mainly through excreta and disposal of unused or expired drugs.
Pharmaceutical compounds are not completely removed during
wastewater treatments and, as a result, they are discharged into
the receiving streams [1,2] and can end up onto soils when sewage
sludge generated is employed as fertilizer [3,4]. The removal of
pharmaceutical compounds in WWTPs is commonly evaluated as
a mass balance between concentrations in inuent and efuent
wastewater [57]. Two processes are mainly responsible for the
removal of pharmaceutical compounds: biodegradation and sorp-
tion onto sludge. An accurate determination of the biodegradation
rates is extremely difcult due to the large number of degradation
products, most of them unknown [4]. Sorption onto sludge depends
on the physical and chemical properties of the compounds and
Corresponding author. Tel.: +34 9 5455 2858; fax: +34 9 5428 2777.
E-mail address: iaparicio@us.es (I. Aparicio).
onto sludge requires experimental studies through the solidwater
partition coefcients (Kd ) [813]. In most cases, Kd values are esti-
mated using batch experiments [8,9]. In other cases, Kd values are
predicted using different equations developed for lipophilic com-
pounds [10,11]. Only a few authors, calculate Kd values from the
measurements of the concentrations of the pharmaceutical com-
pounds in wastewater and sewage sludge [12,13].
The rst aim of this study was to evaluate the occurrence
and distribution of pharmaceutical compounds in wastewater and
sludge lines in wastewater treatment plants. To achieve this aim,
sixteen pharmaceutical compounds belonging to seven therapeu-
tic groups were monitored during 1-year period in inuent and
efuent wastewater and in primary, secondary and digested sludge
from four WWTPs. The second aim was to evaluate the effective-
ness of wastewater and sludge treatment lines to remove these
compounds. Kd values were calculated, from measured concen-
trations in wastewater and sludge, to evaluate the contribution
of sorption onto sludge in the removal. The third aim was to
evaluate the ecotoxicological risk due to efuent wastewater dis-
charges to the receiving streams and to the disposal of sewage
sludge as fertilizer onto soils. Pharmaceutical compounds moni-
tored were ve non-steroidal anti-inammatory drugs (diclofenac,
ibuprofen, ketoprofen, naproxen and salicylic acid), two antibiotics

0304-3894/$ see front matter 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jhazmat.2012.04.068
 J. Martn et al. / Journal of Hazardous Materials 239240 (2012) 4047
(sulfamethoxazole and trimethoprim), a -blocker (propranolol),
two lipid regulators (clobric acid and gembrozil), an antiepileptic
drug (carbamazepine), four estrogens (17-ethinylestradiol, 17-
estradiol, estriol and estrone), and a nervous stimulant (caffeine).
Salicylic acid and clobric acid are metabolites of pharmaceuticals
(acetilsalicylic and clobrate, respectively) extensively metabo-
lized. The presence of caffeine in wastewater is due, in a lower
extent, to its presence in pills and, in a higher extent, to its presence
in food-related sources, as coffee and soft drinks.
2. Experimental
2.1. Chemicals and reagents
HPLC-grade water, acetonitrile and methanol were purchased
from Romil Ltd. (Barcelona, Spain). Hexane, acetone (HPLC
grade) and sulfuric acid of analytical grade were obtained from
Panreac (Barcelona, Spain). Potassium dihydrogen phosphate
of analytical grade was purchased from Scharlau (Barcelona,
Spain). Carbamazepine, diclofenac, ketoprofen, naproxen and sal-
icylic acid (97100% purity) were purchased from SigmaAldrich
(Steinheim, Germany). Caffeine was obtained from Merck (Darm-
stadt, Germany). Clobric acid, gembrozil, ibuprofen, propra-
nolol hydrochloride, 17-ethinylestradiol, 17-estradiol, estriol,
estrone, sulfamethoxazole and trimethoprim (99% purity) were
purchased from Dr. Ehrenstorfer (Augsburg, Germany). Three-
milliliters solid phase extraction (SPE) cartridges, packed with
60 mg of Oasis HLB, were purchased from Waters (Milford, MA,
USA).
Stock solutions of each pharmaceutical compound, at a concen-
tration level of 1000 mg/L, were prepared in methanol and stored at
4 C. Working solutions were prepared by diluting the stock stan-
dard solutions in methanol.
2.2. Wastewater and sludge sampling
Inuent and efuent wastewater and primary, secondary and
digested sludge were sampled from four WWTPs namely north
(350.000 equivalent inhabitants, 62.000 m3 /day), south (950.000
equivalent inhabitants, 164.500 m3 /day), east (200.000 equivalent
inhabitants, 40.900 m3 /day) and west (200.000 equivalent inhabi-
tants, 23.150 m3 /day) WWTP sited in Seville city (south of Spain).
Treatments in the studied WWTPs involve pretreatment, primary
(settling) and secondary (activated sludge) treatments. Fig. 1 shows
a diagram scheme of wastewater and sludge treatment lines with
the sampling points marked in bold. Pretreated wastewater is sub-
jected to settling in primary clariers where primary sludge is
obtained. Secondary treatment is based on activated sludge pro-
cesses in a biologic reactor and settling in secondary clariers
where secondary sludge is produced. Mixed sludge (a mixture of
primary and secondary sludge) is then anaerobically digested and
dehydrated for sludge stabilization.
Sixteen inuent and efuent samples (four samples from each
WWTP) were collected from January 2008 to January 2009 in stud-
ied WWTPs. Daily-composite samples were obtained by mixing the
aliquots collected every hour by an automatic device operating dur-
ing 24 h. Sample volumes collected each hour were proportional
to inuent and efuent ows. A total sample volume of 2.5 L was
transferred to amber glass bottles and stored at 4 C until analysis.
Sixteen sludge samples (four samples from each WWTP) were
collected from each sampling point (Fig. 1). Two liters of primary
and secondary sludge and one kilogram of digested sludge and com-
post were collected in glass bottles. Sludge samples were rstly
decanted and then ltered to remove their water content. Finally,
sludge samples were lyophilized in a Cryodos-50 lyophilizer
41
(Telstar, Terrasa, Spain), sieved (particle size <100 m) and stored,
when necessary, in glass bottles at 30 C until analysis. The water
content of sludge samples after lyophilization was calculated by
drying lyophilized sludge in an oven at 105 C. Their moisture con-
tent was taken into account to refer concentrations to sludge dry
matter.
2.3. Analytical methods
Pharmaceutical compounds were analyzed according to previ-
ously reported and validated methods [14,15]. Extraction of the
pharmaceutically active compounds from wastewater was carried
out by solid-phase extraction according to Camacho-Munoz et al.
[14]. Sludge analysis was based on the extraction of the pharma-
ceutical compounds by ultrasonic-assisted extraction and clean-up
by solid-phase extraction according to Martn et al. [15]. In both
cases, the chromatographic analysis was performed using an HPLC
1200 Series instrument (Agilent, USA) equipped with an ultravio-
let diode array (DAD) and a rapid scan uorescence (Fl) detector
connected on line. Separations were carried out using a Zorbax
Eclipse XDB-C18 (150 mm 4.6 mm, 5 m) cartridge column (Agi-
lent, USA) protected by a XDB-C18 (4 mm 4 mm, 5 m) guard
column (Agilent, USA). Chromatographic analysis was carried out
by gradient elution with acetonitrile and a 25 mM potassium dihy-
drogen phosphate solution as previously reported [14].
2.4. Solidwater partition coefcients, Kd
Solidwater partition coefcients (Kd ), corresponding to the
distributions of the pharmaceutical compounds between primary
sludge and inuent wastewater and between secondary sludge and
efuent wastewater, were calculated by the Eq. (1):
Csolid
Kd =
Cwater
where Kd is expressed in L/kg, Csolid is the concentration of the phar-
maceutical compound in the solid phase (g/kg dry matter (dm))
and Cwater is the concentration of the pharmaceutical compound in
the aqueous phase (g/L).
2.5. Ecotoxicological risk assessment
Ecotoxicological risk assessment was evaluated by means of
risk quotient (RQ) values in efuent wastewater, in the receiving
stream, in digested sludge and in digested-sludge amended soil.
Risk quotient values are usually expressed as the ratio between
the predicted environmental concentration (PEC), or the measured
environmental concentrations (MEC values) when available, and
the predicted no-effect concentration (PNEC). RQ values lower than
1 indicate that the compound does not imply a signicant risk to
the environment.
PECwater values were estimated from the concentration levels
measured (MEC values) in efuent wastewater applying a dilution
factor of 100. The dilution factor was estimated from the ow rates
of efuent wastewater and the receiving stream. PECsoil values,
estimated one year after one sludge-dose application, were calcu-
lated applying the Eq. (2) from the European Commission Technical
Guidance Document on Risk Assessment EUR 20418 EN/2 [16]:
Csludge APPLsludge
PECsoil =
DEPTHsoil RHOsoil
where Csludge is the concentration measured in digested sludge
expressed as g/kg dm; APPLsludge is the application rate of dry-
sludge onto soils (0.5 kg/m2 year for agricultural soils); DEPTHsoil
is the mixing depth (0.20 m for agricultural soils) and RHOsoil is the
bulk density of wet soil (1700 kg/m3 for agricultural soils).
42 J. Martn et al. / Journal of Hazardous Materials 239240 (2012) 4047
Fig. 1. Wastewater and sludge treatment lines in the WWTPs studied.
PNECwater values were calculated from the lowest values of
chronic and acute toxicity data (lethal concentration (LC), effect
concentration (EC) and non-observed effect concentration (NOEC))
reported in literature to several aquatic organisms: bacteria, algae,
invertebrate and sh species [2] by applying an assessment factor of
1000 to consider the toxicity to other aquatic species more sensitive
than those selected in toxicity studies [16,17]. PNECsolid values were
estimated from PNECwater values because no toxicological data of
pharmaceutically active compounds in the terrestrial compartment
was found in literature. PNECsolid values were estimated applying
the equilibrium partition approach [17]:
PNECsolid = PNECwater Kd
where Kd is the solidwater partition coefcient which takes
into account the two main mechanisms of sorption into sludge:
absorption and adsorption [4]. In fate and contaminant transport
calculations, Kd is dened by the United States Environmental
Protection Agency as the ratio of the contaminant concentration
associated with the solid to the contaminant concentration in the
surrounding aqueous solution when the system is at equilibrium.
Kd values from literature [9,12,13,1823] were used (Table 1).
3. Results and discussion
3.1. Occurrence of the pharmaceutically active compounds in
wastewater and sludge
The distribution of the pharmaceutical compounds in wastew-
ater treatment line is shown in Table 2. Gembrozil, ketoprofen,
naproxen, propranolol and salicylic acid were detected in all of the
wastewater samples whereas clobric acid, estrone, sulfamethox-
azole and trimethoprim were not detected in wastewater. The
anti-inammatory drugs were the compounds at the highest con-
centration levels, being ibuprofen and salicylic acid the ones at the
highest concentration levels with mean values, in the four WWTPs
sampled, of 32.0 and 27.2 g/L, respectively, in inuent wastew-
ater and 4.04 and 1.00 g/L, respectively, in efuent wastewater.
Their high concentrations can be related to their wide use enhanced
because no medical prescription is necessary for their sale. In
Spain, the total amount of ibuprofen sold per year has been esti-
mated to be about 276 tons, while antibiotics are sold at a lesser
extent, for example, trimethoprim is being sold at 3.7 tons per year
[24]. The nervous stimulant caffeine, which not only comes from
pharmaceuticals but also from coffee consumption, reached con-
centration levels up to 8.97 g/L, in inuent wastewater, and up to
0.94 g/L in efuent wastewater. The estrogenic compounds were
found at lower concentration with mean values from below the
limits of detection to 0.30 g/L, in inuent wastewater; and from
below the limits of detection to 0.41 g/L, in efuent wastewa-
ter. Alongside wastewater treatment line, the concentrations of
the pharmaceuticals decreased but all the compounds detected in
inuent wastewater were still present in efuent wastewater.
The distribution of the pharmaceutical compounds in sewage
sludge is shown in Table 3. The pattern of occurrence of the
pharmaceutical compounds in sewage sludge was similar to that
observed in wastewater. The highest mean concentrations, in
the four WWTPs sampled, correspond to ibuprofen (2206 g/kg
dm in primary sludge, 1584 g/kg dm in secondary sludge and
1170 g/kg dm in digested sludge). The other compounds at
high concentrations in primary sludge were gembrozil, sali-
cylic acid and caffeine (mean concentration in primary sludge:
873 g/kg dm, 560 g/kg dm and 446 g/kg dm, respectively).
The pharmaceuticals with high log Kow values and low pKa values
as diclofenac, ketoprofen and gembrozil were mainly detected
in wastewater instead of in sludge. At wastewater pH, they are
mainly in their ionized form and, therefore, present in the aqueous
phase.
The pattern of occurrence of the pharmaceutical compounds in
primary sludge was different to that observed in secondary sludge.
For example, the pharmaceutical compounds at the highest con-
centrations in primary sludge were ibuprofen, gembrozil, salicylic
acid and caffeine while, in secondary sludge, the pharmaceutical
compounds at the highest concentrations were carbamazepine,
17-ethinylestradiol, estriol and propranolol. This fact could be
explained by the different physicochemical characteristics of the
studied compounds (Table 4) and the different composition of
primary and secondary sludge, resulting in different sorption
behaviors. After the anaerobic digestion, the concentration levels
of most of the compounds decreased (Table 3). The concentration
 J. Martn et al. / Journal of Hazardous Materials 239240 (2012) 4047 43

Table 1
Partition coefcients and ecotoxicological data of the pharmaceutically active compounds.

Pharmaceutically active Partition coefcient Ecotoxicological data

compound

Log Kd , sludge Log Kd , soil Organism Test Toxicological PNECwater PNECsludge PNECsoil
value (mg/L) (g/L) (g/kg) (g/kg)

Anti-inammatory drugs
Diclofenac 2.70a 2.21b V. scheri (bacteria) EC50 (15 min) 9.70 9.70 4862 1595
Ibuprofen 2.10a 1.45c H. attenuata (invertebrate) EC50 (96 h) 1.65 1.65 1141 256
Ketoprofen 2.40a 0.95d V. scheri (bacteria) EC50 (15 min) 15.6 15.6 3919 140
Naproxen 1.55d 0.95d H. attenuata (invertebrate) EC50 (96 h) 2.62 2.62 752 189
Salicylic acid 1.36d 1.91d V. scheri (bacteria) EC50 (15 min) 43.1 43.1 992 3520
Antibiotics
Sulfametoxazole 1.04d 0.90d P. subcapitata (algae) EC50 (96 h) 0.15 0.15 1.64 1.19
Trimethoprim 1.83d 1.41d D. magna (invertebrate) EC50 (96 h) 121 121 10273 3102
Antiepileptic drug
Carbamazepine 1.15d 1.40d D. magna (invertebrate) EC50 (48 h) 13.8 13.8 195 347
-Blocker
Propranolol 2.52d 1.76d D. subspicatus (algae) EC50 (48 h) 0.70 0.70 232 40.3
Nervous stimulant
Caffeine 2.30a 1.40d Leuciscus Idus (sh) EC50 (96 h) 87.0 87.0 17357 2185
Estrogens
e f
17-Ethinylestradiol 2.46 1.75 S. purpuratus (invertebrate) EC50 0.03 0.03 8.75 1.69
17-Estradiol 2.56e 1.85f S. purpuratus (invertebrate) EC50 0.01 0.01 5.14 0.99
Estriol 2.67g 2.67g S. purpuratus (invertebrate) EC50 1.52 1.52 711 711
Estrone 2.45e 1.40f T. battagliai (invertebrate) LC50 (10 days) 0.10 0.10 28.2 2.51
Lipid regulators
Clobric acid 0.70d 0.95d D. magna (invertebrate) EC50 (48 h) 72.0 72.0 361 642
Gembrozil 1.29h 0.11i H. attenuata (invertebrate) EC50 (96 h) 1.18 1.18 203 13.3
a
Okuda et al. [12].
b
Drillia et al. [18].
c
Stuer-Lauridsen et al. [19].
d
Barron et al. [20].
e
Carballa et al. [9].
f
Sarmah et al. [21].
g
Lpez de Alda et al. [22].
h
Radjenovic et al. [13].
i
Krascsenits et al. [23].

Table 2
Mean concentrations (n = 4) of the analyzed pharmaceutical compounds in inuent and efuent wastewater from each WWTP.

Pharmaceutically active Mean concentration (g/L)

compound

Inuent wastewater Efuent wastewater

North WWTP South WWTP East WWTP West WWTP North WWTP South WWTP East WWTP West WWTP

Anti-inammatory drugs
Diclofenac 0.72 <LOQ0.099 <LOD0.030 <LOD0.030 0.53 0.74 <LOD0.015 0.09
Ibuprofen 50.6 34.0 12.9 25.6 3.74 1.05 3.2 8.00
Ketoprofen 1.69 1.84 2.11 2.08 0.92 0.89 0.88 0.94
Naproxen 4.09 3.84 3.37 2.54 2.58 0.99 1.15 1.30
Salicylic acid 27.8 33.1 12.6 31.7 0.34 0.17 0.10 3.17
Antibiotics
Sulfamethoxazole <LOD0.017 <LOD0.017 <LOD0.017 <LOD0.017 <LOD0.008 <LOD0.008 <LOD0.008 <LOD0.008
Trimethoprim <LOD0.012 <LOD0.012 <LOD0.012 <LOD0.012 <LOD0.006 <LOD0.006 <LOD0.006 <LOD0.006
Antiepileptic drug
Carbamazepine 0.51 0.14 0.07 0.97 0.06 0.15 0.05 0.14
-Blocker
Propranolol 0.39 0.27 0.20 0.23 0.34 0.37 0.21 0.32
Nervous stimulant
Caffeine 0.89 0.44 0.49 3.28 0.24 0.08 0.37 0.30
Estrogens
17-Ethinylestradiol <LOQ0.067 0.15 0.07 0.18 0.12 0.03 0.04 0.18
17-Estradiol <LOQ0.145 0.19 <LOQ0.145 <LOQ0.145 <LOQ0.072 <LOQ0.072 <LOD0.022 <LOQ0.072
Estriol 0.12 0.83 0.10 0.09 0.59 0.27 0.43 0.37
Estrone <LOD0.323 <LOD0.323 <LOD0.323 <LOD0.323 <LOD0.162 <LOD0.162 <LOD0.162 <LOD0.162
Lipid regulators
Clobric acid <LOD0.001 <LOD0.001 <LOD0.001 <LOD0.001 <LOD0.001 <LOD0.001 <LOD0.001 <LOD0.001
Gembrozil 2.64 1.68 1.23 2.69 3.07 2.47 1.52 2.16

LOD: limit of detection; LOQ: limit of quantication. LOD and LOQ superscripts correspond to LOD and LOQ concentration values [14].
44 J. Martn et al. / Journal of Hazardous Materials 239240 (2012) 4047

Table 3
Mean concentrations (n = 4) of the analyzed pharmaceutical compounds in primary, secondary and digested sludge from each WWTP.

Pharmaceutically Mean concentration (g/kg dry matter)

active compound

Primary sludge Secondary sludge Digested sludge

N-WWTP S-WWTP E-WWTP W-WWTP N-WWTP S-WWTP E-WWTP W-WWTP N-WWTP S-WWTP E-WWTP W-WWTP

Anti-inammatory drugs
Diclofenac <LOD33.1 <LOD33.1 <LOD33.1 <LOD33.1 <LOD19.3 <LOD19.3 <LOD19.3 <LOD19.3 <LOD1.22 <LOD1.22 <LOD1.22 <LOD1.22
Ibuprofen 2988 1425 2728 1683 1889 687 3237 524 1020 1274 1262 1124
Ketoprofen <LOD6.18 <LOD6.18 <LOD6.18 <LOD6.18 24.8 <LOD3.79 66.6 24.0 <LOD3.48 <LOD3.48 <LOD3.48 <LOD3.48
Naproxen 40.1 66.6 72.2 23.8 41.4 34.3 32.9 50.4 <LOQ7.53 <LOQ7.53 <LOD2.38 <LOQ7.53
Salicylic acid 389 931 503 419 140 131 51.9 94.3 48.0 14.9 12.8 16.0
Antibiotics
Sulfamethoxazole <LOD8.87 <LOD8.87 <LOD8.87 <LOD8.87 <LOD10.4 <LOD10.4 <LOD10.4 <LOD10.4 <LOD47.2 <LOD47.2 <LOD47.2 <LOD47.2
Trimethoprim <LOD53.2 <LOD53.2 <LOD53.2 <LOD53.2 <LOD81.8 <LOD81.8 <LOD81.8 <LOD81.8 <LOD24.6 <LOD24.6 <LOD24.6 <LOD24.6
Antiepileptic drug
Carbamazepine 20.3 30.3 <LOQ14.8 66.6 259 262 231 460 28.4 18.5 30.8 18.4
-Blocker
Propranolol <LOD1.60 <LOD1.60 <LOD1.60 <LOD1.60 8.58 13.9 32.5 26.6 <LOD1.26 <LOQ2.51 <LOD1.26 <LOD1.26
Nervous stimulant
Caffeine 674 527 401 183 <LOD20.4 <LOD20.4 <LOD20.4 <LOD20.4 <LOQ45.4 <LOQ45.4 <LOQ45.4 116
Estrogens
17- 46.8 28.5 33.0 52.7 105 103 23.8 160 56.8 70.5 19.8 60.2
Ethinylestradiol
17-Estradiol 17.1 7.69 11.4 25.4 20.5 23.8 16.6 38.0 836 120 116 102
Estriol 8.53 7.93 4.03 12.3 <LOD3.09 60.8 68.0 23.4 35.2 4.03 2.18 4.62
Estrone <LOD10.1 <LOD10.1 <LOD10.1 <LOD10.1 <LOD7.68 <LOD7.68 <LOD7.68 <LOD7.68 <LOD4.95 <LOD4.95 <LOD4.95 <LOD4.95
Lipid regulators
Clobric acid <LOD32.9 <LOD32.9 <LOD32.9 <LOD32.9 <LOD36.4 <LOD36.4 <LOD36.4 <LOD36.4 <LOD38.1 <LOD38.1 <LOD38.1 <LOD38.1
Gembrozil 1099 <LOQ360 2026 <LOQ360 <LOQ350 356 <LOQ350 <LOQ350 <LOD79.8 <LOD79.8 <LOD79.8 <LOD79.8

N-WWTP: north WWTP, S-WWTP: south WWTP, E-WWTP: east WWTP, W-WWTP: west WWTP.
LOD and LOQ superscripts correspond to LOD and LOQ concentration values.

of 17-estradiol increased after digestion, mean values, in the four
WWTPs sampled, from 15 g/kg dm in primary sludge to 294 g/kg
dm in digested sludge. This fact could be explained by the cleavage
of conjugated steroid estrogens [2527] and by the concentra-
tion that undergo persistent pollutants due to the loss of weight
of sludge after digestion process. There is also a possibility of
pharmaceutical compounds to enter the food chain being trans-
ferred from soil to plants via root systems when wastewater is
employed to irrigate agricultural soils or sludge is used as fertil-
izer. Such uptake by plants has been reported for carbamazepine
[28,29], sulfamethoxazole [28], trimetroprim [28], ibuprofen [30]
and 17-ethynilestradiol [31].

Table 4
Physicochemical properties (log Kow and pKa values) and calculated log Kd values of the pharmaceutically active compounds.

Pharmaceutically Physicochemical properties Calculated log Kd values

active compound

log Kow pKa Primary sludge Secondary sludge

N-WWTP S-WWTP E-WWTP W-WWTP N-WWTP S-WWTP E-WWTP W-WWTP

Anti-inammatory drugs
Diclofenac 4.8 4.1
Ibuprofen 4.5 4.95.7 1.77 1.62 2.33 1.82 2.70 2.82 3.01 1.82
Ketoprofen 4.0 4.55.7 1.43 1.88 1.41
Naproxen 3.2 4.2 0.99 1.24 1.33 0.97 1.21 1.54 1.46 1.59
Salicylic acid 2.3 3.5 1.15 1.45 1.60 1.12 2.61 2.89 2.72 1.47
Antibiotics
Sulfamethoxazole 0.9 5.66.7
Trimethoprim 1.4 6.6
Antiepileptic drug
Carbamazepine 2.4 13.9 1.60 2.34 1.84 3.64 3.24 3.66 3.52
-Blocker
Propranolol 0.7 9.5 1.40 1.57 2.19 1.92
Nervous stimulant
Caffeine 0.1 10.4 2.88 3.08 2.91 1.75
Estrogens
17- 4.2 10.410.7 2.28 2.67 2.47 2.94 3.54 2.77 2.95
Ethinylestradiol
17-Estradiol 4.0 10.4 1.61
Estriol 2.8 9.8 1.85 0.98 1.61 2.14 2.35 2.20 1.80
Estrone 4.1 10.4
Lipid regulators
Clobric acid 2.6 2.53.2
Gembrozil 4.8 4.7 2.62 3.22 2.16

: Compound not detected; N-WWTP: north WWTP; S-WWTP: south WWTP; E-WWTP: east WWTP; W-WWTP: west WWTP.
 J. Martn et al. / Journal of Hazardous Materials 239240 (2012) 4047
Fig. 2. Mean removal efciencies (%) of the investigated pharmaceutical compounds. Lines in each bar show maximum and minimum removal values. (Aep: antiepileptic
drug; -Bl: -blocker; Ns: nervous stimulant; Lrg: lipid regulator.)
3.2. Solidwater partition coefcients, Kd
Kd values, in primary sludge and secondary sludge, calculated
from experimental concentrations measured in wastewater and
sludge are shown in Table 4. Log Kd values in secondary sludge were
higher than those in primary sludge. This fact, depending on the
physicochemical properties of the compound (log Kow and pKa val-
ues), can be due to a better sorption onto secondary sludge than
onto primary sludge or to a fast biodegradation. The acidic com-
pounds, characterized by low pKa values, are expected to have low
log Kd values because at wastewater pH they are mainly in their
ionized form and, consequently, mainly dissolved in the aqueous
phase. Nevertheless, log Kd values of the anti-inammatory drugs,
all of them with carboxylic groups, are just slightly lower, in pri-
mary sludge, and similar, in secondary sludge, than those of the
other pharmaceutical compounds with no carboxylic group. The
unexpectedly high log Kd values of the anti-inammatory drugs
cannot be associated to a high potential of sorption onto sludge
but to a fast biodegradation that reduce the measured concentra-
tions in the aqueous phase. The same phenomenon was reported,
for the anti-inammatory drug acetaminophen, by Radjenovic et al.
[13]. The high log Kd values of carbamazepine, 17-ethinylestradiol
and estriol indicate that the compounds tend to be retained onto
sludge which is consistent with their high pKa values. This fact is in
agreement with that reported in the literature [8,9,20,32].
3.3. Removal of pharmaceutically active compounds from
wastewater
The removal rates achieved in each WWTP and the average
removal rates in the four WWTPs evaluated are shown in Fig. 2.
Signicant variation was observed from one compound to another
but no signicant different removal behavior was observed among
the four WWTPs evaluated. The main mechanisms of removal
of pharmaceutical compounds from wastewater are biodegrada-
tion or sorption onto sludge. The anti-inammatory drugs salicylic
acid and ibuprofen were the best removed pharmaceutical com-
pounds (mean removal rates: 99 and 87%, respectively). Both
compounds have low pKa values (Table 4) so they are expected
45
to be mainly in the aqueous phase. Their removal from wastew-
ater could be explained by biodegradation instead of by sorption
onto sludge. This fact is consistent with that reported by sev-
eral authors [6,33,34] who propose biodegradation as the most
probable mechanism for their removal. Diclofenac was the anti-
inammatory drug poorest removed, mean removal rate: 14%. The
poor removal of diclofenac can be probably due to the combination
of degradation in wastewater together with the liberation of addi-
tional diclofenac molecules by de-conjugation of glucoronidated
or sulfated diclofenac and/or its desorption from particles [7].
Similar poor removal rates (<16%) were observed for the lipid reg-
ulator gembrozil and the -blocker propranolol. Carbamazepine
is a pharmaceutical compound with a high chemical stability to
the point that has been proposed as a anthropogenic marker
[5,6]. Therefore, the removal of carbamazepine from wastewater
could be explained by sorption onto sludge due to its hydrophilic
nature (log Kow : 2.4). Elimination rates of caffeine vary signi-
cantly, not only from one WWTP to another, but also in the same
WWTP at different time periods what can be associated to its
temporal-conditioned consume. The low polarity of the estrogenic
compounds, characterized by log Kow values between 2.8 and 4.2
(Table 4), makes sorption onto sludge to be the most likely process
responsible for their removal from wastewater (17-estradiol: 55%,
17-ethinylestradiol: 47% and estriol: 26%) [35].
3.4. Risk assessment
Fig. 3 shows RQ values of each pharmaceutical compound in
four different scenarios: efuent wastewater, receiving streams,
digested sludge and digested-sludge amended soil. To estimate
RQ values in efuent wastewater and digested sludge, the high-
est concentration levels measured in efuent wastewater and
digested sludge were used as MEC values. To estimate RQ values
in receiving streams and in digested-sludge amended soils, the
highest PEC values were applied. The compounds responsible
for the highest environmental risks in efuent wastewater and
digested sludge were 17-estradiol (RQ values 12 and 359, respec-
tively), 17-ethinylestradiol (RQ values 12 and 22, respectively)
and ibuprofen (RQ values 3.2 and 4.4, respectively). RQ values of
46 J. Martn et al. / Journal of Hazardous Materials 239240 (2012) 4047
Fig. 3. Risk quotient (RQ) values of the pharmaceutical compounds in efuent wastewater, the receiving stream, digested sludge and digested-sludge amended soil. (Aep:
antiepileptic drug; -Bl: -blocker; Ns: nervous stimulant; Lrg: lipid regulator.)
the other pharmaceutical compounds varied between 0.01 and 0.7
in efuent wastewater and 0.020.26 in digested sludge so no risk
is suspected to occur in these two scenarios.
The dilution effect that takes place after the discharge of efuent
wastewater into the receiving stream results in a decrease of the
concentrations of the pharmaceutical compounds. The concentra-
tions of all the pharmaceutical compounds in the receiving stream
were low enough that no risk to aquatic organisms is suspected
to occur. All the RQ values were lower than 1 (Fig. 3). Therefore,
acute toxic effect in the aquatic environment, with the current use
of pharmaceutical compounds, is unlikely. However, toxic effects
due to chronic environmental exposure, mainly by aquatic species
with a long-life cycle, could still be present. In a previous paper [2],
the occurrence and risk assessment of the presence of the phar-
maceutical compounds in Guadalquivir River were reported. The
results provided in the present paper shows that the low RQ val-
ues estimated in the previous paper are due to the dilution effect
of wastewater discharges into the receiving stream. Regarding to
sludge samples, after sludge application onto soil, a drastic decrease
of RQ values was observed. The only toxicological effect expected is
the one caused by 17-estradiol since the RQ value of 17-estradiol
exceeds the limit value of 1 for the most sensitive species (RQ value:
2.7). This fact means that an ecotoxicological risk is still present
to terrestrial ecosystems in spite of the important decrease of the
concentration of 17-estradiol from digested sludge to digested-
sludge amended soil.
4. Conclusions
The highest concentrations in wastewater and sludge were
observed for the anti-inammatory drugs ibuprofen and salicylic
acid. The pharmaceutical compounds detected in wastewater were
also detected in sludge, except diclofenac which was only detected
in wastewater. In wastewater treatment line, the most persistent
compounds were diclofenac, propranolol, estriol and gembrozil.
The best removed compounds were ibuprofen and salycilic acid. In
sludge line, different behavior of the pharmaceutical compounds
was observed as well. The concentrations of some compounds
as naproxen and salicylic acid decrease from primary sludge to
digested sludge; the concentrations of other compounds as 17-
estradiol, increase; and the concentration of the other compounds
as 17-ethinylestradiol and estriol, do not vary signicantly. The
concentrations measured in sludge indicate that sorption can be
one of the key factors controlling the removal of pharmaceutical
compounds. The removal of the anti-inammatory drugs and the
lipid regulator gembrozil cannot be explained by sorption onto
sludge but by biodegradation in the aqueous phase. Environmental
risk assessment revealed a high ecotoxicological risk due to ibupro-
fen, 17-ethinylestradiol and 17-estradiol both for aquatic and
terrestrial ecosystems. After disposal of wastewater in the receiv-
ing stream and sludge disposal onto soils, a signicant decrease
of the ecotoxicological risk occurs. Nevertheless, RQ value of 17-
estradiol in digested-sludge amended soil is high enough to cause
ecotoxicological risk to the most sensitive species in the terrestrial
ecosystem. The results obtained shown the necessity to improve
wastewater treatments to reduce the input of pharmaceutical com-
pounds in surface water and soils. Special attention should be paid
to the presence of ibuprofen and the estrogenic compounds in
digested sludge mainly when sludge is used as fertilizer onto agri-
cultural soils because they could even enter in the food chain after
the uptake of the pharmaceuticals by plants.
Acknowledgements
The authors wish to thank the nancial support received
from the Ministerio de Educacin y Ciencia, Spain (project no.
CGL2007-62281) and from the Programa de Becas de Formacin
de Profesorado Universitario (Ministerio de Educacin, Spain).
 J. Martn et al. / Journal of Hazardous Materials 239240 (2012) 4047
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