Pharmaceuticals in the environment in Italy: causes, occurrence, effects and control.

Abstract

Environmental contamination by pharmaceuticals is an emerging issue. Until recently, information on medicinal substances released into the environment was scant, but several studies have now been published. Data are, however, usually scattered and a systematic approach to this subject is generally lacking. Moreover, because of differences in the prevalence of diseases, treatment habits and options, or simply for market reasons, the pollution profile can differ significantly in different countries. The aim of this work is to review the papers dealing with environmental contamination by pharmaceuticals in Italy, with the aim of providing a comprehensive view on a national scale.
Papers related to environmental contamination by pharmaceuticals in Italy were reviewed, in order to offer a comprehensive view of this subject. Topics included analysis, occurrence, monitoring, modelling, treatment, control of the emissions, and ecotoxicological effects of pharmaceuticals in the environment.
The literature suggests that pharmaceuticals are widespread contaminants, entering the environment from a myriad of scattered points. Patients, in case of drugs for human use, or animals for veterinary drugs, are the main sources of contamination. Pharmaceuticals can be ranked according to environmental loads, predicted by multiplying sales figures by the rate of metabolism in man or animals. Priority pharmaceuticals, i.e. the molecules of concern for the environment, can be measured in waste and surface water by liquid chromatography-tandem mass spectrometry, and the loads detected are generally comparable to the predicted ones. Pharmaceuticals are designed to stimulate a response in humans and animals at low doses, with a very specific target, so the implications for human health and the environment need to be assessed. In vitro and in vivo studies suggest that pharmaceutical principles, taken singularly or in combinations, and concentrations close to those detected in the environment, may have ecotoxicological effects. The sewage system is an important point in the control of contamination, but sewage treatment plants are not able efficiently to abate a substantial part of water-borne pharmaceuticals. Several variables play a role, however, in the processes of waste water treatment, and could be specifically adjusted to improve the efficiency of drug abatement, mitigating the potential environmental hazards.
Pharmaceuticals in the environment are becoming a subject of global concern, with potential environmental consequences. Further knowledge of the causes, occurrence and effects of drugs as environmental pollutants is necessary for a better understanding of this ecological issue, as well as to improve abatement strategies, and to mitigate subtle environmental consequences.

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Review ArticlesPharmaceuticals in the Environment in Italy
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© 2006 ecomed publishers (Verlagsgruppe Hüthig Jehle Rehm GmbH), D-86899 Landsberg and Tokyo • Mumbai • Seoul • Melbourne • Paris
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Review Articles
Pharmaceuticals in the Environment in Italy: Causes, Occurrence,
Effects and Control
Ettore Zuccato1*, Sara Castiglioni1/2, Roberto Fanelli1, Giuseppe Reitano1, Renzo Bagnati1, Chiara Chiabrando1,
Francesco Pomati2, Carlo Rossetti2 and Davide Calamari2
1Department of Environmental Health Sciences, Mario Negri Institute for Pharmacological Research, via Eritrea 62, 20157 Milan, Italy
2Department of Biotechnology and Molecular Sciences, University of Insubria, Via J.H. Dunant 3, 21100 Varese, Italy
* Corresponding author (zuccato@marionegri.it)
In memory of Davide Calamari
Introduction
Pharmaceuticals in the environment are a recent issue. Some
preliminary data were already published in 1976 in USA
and in 1985 in England but systematic research started only
in the '90s when in Germany some results were published of
monitoring studies measuring pharmaceuticals in local riv-
ers and sewage treatment plants (STPs). In 1996 we started
monitoring in Italy, so far almost ten years now our group,
made up by scientists from the Insubria University in Varese
led by Davide Calamari, and from Istituto Mario Negri in
Milan, has been working on this topic. In this period several
studies have been completed and several papers published,
on analysis, occurrence, monitoring, modelling, treatment,
control of emissions, and ecotoxicological effects of phar-
maceuticals in the environment. This paper reviews this
work, to depict a view of this environmental issue in Italy.
The paper is also intended as a tribute to Davide Calamari,
co-author of the most of the papers cited in this review. We
dedicate this paper to his memory.
1
Pharmaceuticals are widespread contaminants, entering the
environment from a myriad of scattered points. Patients, in
case of drugs for human use, or animals for veterinary drugs,
are the main sources of contamination. Pharmaceuticals can
be excreted as the parent compound and/or metabolites in
urine and feces. Several pharmaceuticals widely used in hu-
man medicine are excreted unchanged or as active metabo-
lites in high percentages, and continuously discharged into
domestic waste waters (Kummerer 2004a). Unwanted or
expired medications are often improperly disposed of, di-
rectly in wastewater. Several pharmaceuticals can therefore
reach STPs in substantial amounts and, if they escape deg-
radation, can be released into surface water. Most wastewa-
ter biological treatments do not completely remove thera-
peutic compounds, which flow into surface waters, and
eventually into ground waters (Ternes 1998, Stumpf et al.
1999). Recent analytical studies in Italy confirm that sev-
eral pharmaceuticals are poorly removed in STPs (Castiglioni
et al. 2004a). Moreover, some pharmaceuticals can be ex-
creted as conjugates, and may release the active moiety by
cleavage during treatment in STPs (Heberer 2002). In case
of drugs for human use, STPs might therefore be considered
an important point source of contamination. Besides im-
Origin and Causes of Contamination
DOI: http://dx.doi.org/10.1065/espr2006.01.004
Abstract
Background, Aim and Scope. Environmental contamination by phar-
maceuticals is an emerging issue. Until recently, information on me-
dicinal substances released into the environment was scant, but sev-
eral studies have now been published. Data are, however, usually
scattered and a systematic approach to this subject is generally lack-
ing. Moreover, because of differences in the prevalence of diseases,
treatment habits and options, or simply for market reasons, the pol-
lution profile can differ significantly in different countries. The aim
of this work is to review the papers dealing with environmental con-
tamination by pharmaceuticals in Italy, with the aim of providing a
comprehensive view on a national scale.
Methods. Papers related to environmental contamination by phar-
maceuticals in Italy were reviewed, in order to offer a comprehen-
sive view of this subject. Topics included analysis, occurrence, moni-
toring, modelling, treatment, control of the emissions, and eco-
toxicological effects of pharmaceuticals in the environment.
Results and Conclusion. The literature suggests that pharmaceuti-
cals are widespread contaminants, entering the environment from a
myriad of scattered points. Patients, in case of drugs for human use,
or animals for veterinary drugs, are the main sources of contamina-
tion. Pharmaceuticals can be ranked according to environmental
loads, predicted by multiplying sales figures by the rate of metabo-
lism in man or animals. Priority pharmaceuticals, i.e. the molecules
of concern for the environment, can be measured in waste and sur-
face water by liquid chromatography-tandem mass spectrometry,
and the loads detected are generally comparable to the predicted
ones. Pharmaceuticals are designed to stimulate a response in hu-
mans and animals at low doses, with a very specific target, so the
implications for human health and the environment need to be as-
sessed. In vitro and in vivo studies suggest that pharmaceutical prin-
ciples, taken singularly or in combinations, and concentrations close
to those detected in the environment, may have ecotoxicological ef-
fects. The sewage system is an important point in the control of
contamination, but sewage treatment plants are not able efficiently
to abate a substantial part of water-borne pharmaceuticals. Several
variables play a role, however, in the processes of waste water treat-
ment, and could be specifically adjusted to improve the efficiency of
drug abatement, mitigating the potential environmental hazards.
Recommendation and Perspective. Pharmaceuticals in the environ-
ment are becoming a subject of global concern, with potential envi-
ronmental consequences. Further knowledge of the causes, occur-
rence and effects of drugs as environmental pollutants is necessary
for a better understanding of this ecological issue, as well as to
improve abatement strategies, and to mitigate subtle environmen-
tal consequences.
Keywords: Analysis; ecotoxicology; environment, Italy; pharmaceu-
ticals; sewage treatment plant; surface water; waste water

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proper disposal of expired medications, manufacturing fa-
cilities are other important local point sources (Zuccato et
al. 2000). Aquaculture industries and run-off from farms
play an additional significant role in the presence of drugs
in the environment (Calamari et al. 2003). Since pharma-
ceuticals may have long half-lives in the environment, they
can accumulate, reaching detectable and biologically active
levels. The environmental persistence of several commonly
used medicinal drugs, such as erythromycin, cyclophospha-
mide, naproxen, sulphamethoxazole, or sulphasalazine, is
longer than one year. Clofibric acid, the main metabolite of
clofibrate, is still detectable in rivers years after its with-
drawal from the market (Zuccato et al. 2000). Several phar-
maceuticals are consequently detectable in surface waters (riv-
ers, lakes and seas) in the ng/L up to the µg/L range (Calamari
et al. 2003, Zuccato 2004, Castiglioni et al. 2004a). The
recent literature is therefore rich in studies on levels of phar-
maceuticals in environmental media. However, thousands of
different pharmaceuticals are currently used in Italy and world-
wide, with hundreds of new molecules synthesized every year
to replace obsolete compounds. Moreover because of differ-
ences in the prevalence of diseases, treatment habits and op-
tions, or simply for market reasons, the pollution profile can
vary significantly among different countries. Contamination
by pharmaceuticals is therefore a variable and changing is-
sue, needing continuous monitoring.
2
Environmental contamination by pharmaceuticals needs to be
monitored for several reasons, including reliable assessment
of risks for the environment and, through the food chain, for
man. However, thousands of different pharmaceuticals are
currently used so blanket monitoring is unthinkable because
of the excessive number of drugs and metabolites, with differ-
ent chemical structures and physico-chemical properties. It is
therefore best to focus on molecules of concern for the envi-
ronment and to establish priorities so as to restrict studies to a
limited number of hazardous molecules. The first step is there-
fore a preliminary selection, to identify a restricted list of phar-
maceuticals most likely to cause environmental problems. The
recent literature provides some examples. A method used in the
USA for preliminary screening took into account the number of
prescriptions and sales figures (Erickson 2002). Another method
also considered the wastewater pathways, the human and envi-
ronmental implications, the representativity of certain classes
of compounds or sources and the availability of analytical meth-
ods to detect the compounds (Kolpin et al. 2002). In Ger-
many, analysis was limited to pharmaceuticals produced in
amounts over ten tons/year, for which analytical methods and
reference standards were available (Erickson 2002).
In Italy we identified a group of potential contaminants, se-
lected on the basis of their mass balance and theoretical loads,
i.e. multiplying sales by percentages of metabolic excretion in
humans (Zuccato et al. 2000). This provided a list of 'top'
pharmaceuticals for human use, excreted unmetabolized in
substantial amounts, with estimated environmental loads in
the order of tons per year (amoxycillin, atenolol, ranitidine,
ceftazidime, lincomycin, erythromycin, ceftriaxone, furo-
semide, salbutamol). A second group of pharmaceuticals was
selected because they had already been measured in the envi-
Prioritisation of the Pharmaceuticals
ronment in Europe in previous occasional surveys (ibuprofen,
diazepam, bezafibrate, cyclophosphamide, clofibric acid), while
a third group consisted of drugs used in Italy as growth pro-
moters (tylosin, tilmicosin, oleandomycin, spiramycin), which
were added to the list to check environmental contamina-
tion from veterinary drugs. In this work the top pharmaceu-
ticals for human use were selected on the basis of their an-
nual sales in Italy, estimated from figures provided by the
pharmaceutical industry. In a subsequent study, the list was
revised to take account of changes in the pharmaceutical
market and sales were calculated from official prescription
figures, from the Ministry of Health or other administrative
bodies (Calamari et al. 2003). However, several new mol-
ecules are synthesized every year by the pharmaceutical in-
dustry, so contamination is a changing issue, needing con-
tinuous updating. The list was therefore updated during
further studies (Castiglioni et al. 2004b, Zuccato et al. 2004)
and improved by including some molecules with high activ-
ity and potential toxicity, in spite of low tonnage, like estro-
gens and anti-cancer drugs, two metabolites (clofibric acid,
the main metabolite of clofibrate, and demethyl diazepam,
a metabolite of diazepam) and the natural estrogens 17β-
estradiol and estrone, which were included in the list to study
total estrogenic activity. Table 1 show the updated list of
'priority pharmaceuticals' for monitoring and ecotoxicology
studies, as recently published (Castiglioni et al. 2005a).
3
In the last ten years several analytical methods have been
published to measure pharmaceuticals in STPs (Ternes 1998,
Stumpf et al. 1999, Metcalfe et al. 2003), rivers (Heberer
2002, Kolpin et al. 2002), the sea (Buser et al. 1998), lakes
(Tixier et al. 2003) and groundwater (Sacher et al. 2001).
Some methods were intended for the detection of specific
therapeutic categories (Miao et al. 2002, Kuch and Ball-
schmiter 2001, Hirsch et al. 1998, Zuehlke et al. 2004),
while others measured wide ranges of compounds (Hernando
et al. 2004, Cahill et al. 2004, Quintana et al. 2004). A
Analysis

Penicillins
Quinolones
Macrolides-lincosamides
Amoxycillin
Ciprofloxacin, ofloxacin
Clarithromycin, erythromycin,
lincomycin, spiramycin
Sulphamethoxazole
Furosemide, hydrochlorothiazide
Atenolol, enalapril
Omeprazole, ranitidine
Carbamazepine, diazepam
Ibuprofen
Salbutamol
Bezafibrate
Ethinylestradiol
Cyclophosphamide, methotrexate
Oleandomycin, tilmicosin, tylosin,
oxytetracycline
17β-estradiol, estrone
Clofibric acid, demethyl-diazepam
Sulfamides
Diuretics
Cardiovascular
Gastrointestinal
CNS drugs
Anti-inflammatory
Bronchodilators
Lipid regulator
Estrogens
Antitumorals
Veterinary antibiotics
Natural estrogens
Metabolites


Table 1: List of ('priority pharmaceuticals' for monitoring and ecotoxicology
studies (Castiglioni et al. 2005a)

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specific analytical multiresidue method for the simultaneous
determination of 30 pharmaceuticals belonging to various
therapeutic categories in waste water has been recently vali-
dated in Italy (Castiglioni et al. 2005a). It is an improve-
ment of two other methods developed to measure pharma-
ceuticals in drinking and surface water and sediment (Zuc-
cato et al. 2000, Calamari et al. 2003). Water samples were
extracted using solid phase extraction cartridges (SPE), and
sediments were extracted using a specific solvent in an ul-
trasonic bath. Before analysis, samples were filtered (glass
microfiber filters GF/D 2.7 µm, Whatman, Kent, UK) and
then processed through different SPE cartridges. Samples
were subsequently analysed by high-pressure liquid-chro-
matography tandem mass spectrometry (HPLC–MS-MS). To
optimise the extraction method, in preliminary work sev-
eral solid phase extraction cartridges were tested, with vari-
ous pH and elution conditions. This led to the selection of
two cartridges, Oasis MCX (60 mg, Waters Corp., Milford,
MA) at pH 1.5–2.0 for the extraction of a first group of
pharmaceuticals, and Lichrolut EN at pH 7.0 (200 mg,
Merck, Darmstadt, Germany) for a second group.
For 'group 1', 500 mL water samples were spiked with 10 ng
of internal standards; 500 mg of Na4-EDTA were added to
prevent tetracyclines complexing with Ca2+ and Mg2+ ions
and residual metals on the SPE cartridges. The pH was then
adjusted to 2.0 with 37% HCl. The Oasis MCX cartridges
were conditioned before use with 6 mL methanol, 3 mL Milli-Q
water and 3 mL water acidified to pH 2, and eluted with 2 mL
methanol, 2 mL 2% ammonia solution in methanol and 2 mL
0.2% sodium hydroxide in methanol. For 'group 2', 500 mL
aqueous samples were adjusted to pH 7.0 with 30% am-
monium hydroxide and spiked with 10 ng of internal stan-
dards. The cartridges were conditioned before use with 6 mL
methanol and 6 mL Milli-Q water and eluted with 3 mL metha-
nol and 3 mL ethyl acetate. The eluates were dried and redis-
solved in 100 µL of acetic acid 0.01% in Milli-Q water (pH
3.5). All the pharmaceuticals were analysed by HPLC-MS-
MS, while in previous studies, some pharmaceuticals, namely
clofibric acid and ibuprofen, were analyzed by GC-MS-MS
(Zuccato et al. 2000). The HPLC system consisted of two
Perkin-Elmer Series 200 pumps and a Perkin-Elmer Series 200
auto sampler. A Luna C8 column 50 mm × 2 mm i.d., 3 µm
particle size (Phenomenex, Torrance, CA, USA) was used for
the chromatographic separation. An API 3000 triple quadru-
pole (Q1q2Q3) mass spectrometer equipped with a turbo ion
spray source (Applied Biosystems – Sciex, Thornhill, Ontario,
Canada) was used in both the positive and negative ionisation
modes. Mass spectrometric analyses were done in the mul-
tiple reaction monitoring (MRM) mode, measuring the frag-
mentation products of the protonated or deprotonated pseudo-
molecular ions of each drug and internal standard (I.S.). For
quantification we used three deuterated internal standards:
salbutamol-D3, to quantify the pharmaceuticals analyzed in
the positive ion mode, ibuprofen-D3 and 17 β-estradiol-D2 for
pharmaceuticals analyzed in the negative ion mode.
Recoveries of the pharmaceuticals were mostly more than
70% and the overall variability of the method was below
8%. The limits of quantification (LOQ) were in the low ng/L
range. The linearity range was between 0.1 and 3000 ng/
100 µL (10 µL injected) and the correlation coefficients were
mostly higher than 0.997. By this method, pharmaceuticals
were normally detected in surface and drinking water in the
ng/L range and in sediment in the ng/kg range.
4 Pharmaceuticals in the Environment
4.1Occurrence of pharmaceuticals in surface, waste, drinking
water and sediments
Except for two preliminary studies that measured levels of
pharmaceuticals in the environment, published in 1977
(Hignite & Azarnoff 1977) and in 1985 (Richardson &
Bowron 1985), the first work with a systematic approach
started to appear in the 1990s, dealing with levels of phar-
maceuticals in local rivers and STPs in Germany (Ternes
1998, Hirsch et al. 1999). The presence of several pharma-
ceuticals in STP effluents was soon confirmed in The Neth-
erlands (Belfroid et al. 1999), Switzerland (Golet et al. 2001),
United Kingdom (Johnson & Sumpter 2001), France, Greece,
and Sweden (Andreozzi et al. 2003), Spain (Carballa et al.
2004) USA (Kolpin et al. 2002, Huggett et al. 2003, Yang
& Carlson 2004), Canada (Metcalfe et al. 2003), Brazil
(Stumpf et al. 1999) and Australia (Braga et al. 2004).
In Italy a first paper (Zuccato et al. 2000), showed there
were several 'priority' pharmaceuticals in water and sedi-
ments from the rivers Po, Lambro and Adda. Drinking wa-
ter samples from the mains water systems showed traces of
diazepam, clofibric acid and tylosin in samples from the cit-
ies of Lodi and Varese. Traces of some pharmaceuticals were
also present in sediments from the same rivers. The wide-
spread occurrence of pharmaceuticals in the Italian envi-
ronment was subsequently confirmed by the same authors
(Calamari et al. 2003) and by others (Andreozzi et al. 2003).
This paper (Calamari et al. 2003) reports results of an ex-
tensive investigation with eight sampling stations along the
rivers Po and Lambro, from which we plotted the patterns
of contamination in a highly populated region with a large
number of animal farms. In the first study, 13 pharmaceuti-
cals for human or veterinary use were detected in the 0.1–
250 ng/L range, but in this second campaign the method
was revised, adding new compounds, selected as previously
described, so we could detect 18 pharmaceuticals in signifi-
cant amounts. Further studies subsequently measured phar-
maceuticals for human use in the influents and effluents of
urban STPs. Effluents of nine STPs spread over Italy, namely
Cagliari, Latina, Cuneo, Varese, Cosenza, Palermo, Naples
and Monza, were analysed, and several pharmaceuticals were
found in the microgram range. By comparing STP levels with
those previously measured in the Po and Lambro rivers
(Table 2), it was possible to identify a group of pollutants
important for the environment in Northern Italy, namely
ofloxacin, furosemide, atenolol, hydrochlorothiazide,
carbamazepine, ibuprofen, spiramycin, bezafibrate, eryth-
romycin, lincomycin and clarithromycin (Zuccato et al.
2005a). These pharmaceuticals were detected in high con-
centrations in STP effluents and were persistent enough to
remain in substantial amounts in river waters too.
4.2
During their survey of therapeutic drugs in environmental
waters, Calamari et al. (2003) found strikingly high levels
Cocaine and illicit drugs in surface and waste waters

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Table 2: Pharmaceuticals in urban sewage treatment plants (median of nine STPs), River Po (median and maximum value of seven sampling sites), and
River Lambro (from: Zuccato et al. 2005a)

ng/L STPs Lambro River
Median
Ofloxacin 600.0 306.1
Furosemide 585.0 254.7
Atenolol 466.0 241.0
Hydrochlorothiazide 439.1 255.8
Carbamazepine 291.1 175.3
Ranitidine 288.2 38.5
Ciprofloxacin 251.0 14.4
Sulphamethoxazole 127.2
Ibuprofen 121.2 20.0
Spiramycin 75.0 74.2
Bezafibrate 54.8 57.2
Erythromycin 47.4 4.5
Lincomycin 30.5 24.4
Clarithromycin 18.1 8.3
Salbutamol 8.7 2.5
Amoxycillin 4.7
Cyclophosphamide 0.6
Diazepam 0.0
Enalapril 0.0 0.5
Ethinylestradiol 0.0
Methotrexate 0.0
Omeprazole 0.0

of a bronchodilator drug, salbutamol, at a certain sampling
site in the River Po. In contrast with most other therapeutic
drugs, these levels could not be explained by local prescrip-
tion figures. It is known that salbutamol is sometimes used
illegally as an anabolizing agent in farm animals, and the
area under study was rich with farms sending sewage into
the river. Therefore, Zuccato and colleagues started to won-
der whether other illegally used or illicit drugs could be
tracked in the river and urban sewage, and whether their
levels could be used to estimate local drug abuse, given that
current methods are indirect and possibly biased. Christian
Daughton of the US Environmental Protection Agency had
in the meantime proposed the use of drug levels in sewage
to monitor drug abuse (Daughton 2001), but their presence
had not yet been proved nor his idea implemented. Zuccato
et al. (2005b) chose cocaine as the first drug to seek and
measure in environmental water samples. Cocaine and its
major urinary metabolite (benzoylecgonine, BE) was indeed
identified by mass spectrometry in all waste water samples
tested and in the Po (the largest Italian river). The river car-
ried the equivalent of 4 kg of cocaine per day. Using the
levels of BE to estimate the corresponding loads of cocaine,
these authors found that the local drug consumption was
considerably higher than current estimates. This quantita-
tive approach – in principle extendable to other illicit drugs
– was therefore proposed as an additional tool to estimate
drug abuse better in real time (Zuccato et al. 2005b).
5
Environmental pollution by pharmaceuticals is a complex
issue, involving thousands of different active molecules, be-
Predictive Approaches
longing to various therapeutic classes, with different physico-
chemical properties, chemical structures, environmental
behaviour and persistence. Blanket monitoring would be
difficult because of the huge number of drugs and metabo-
lites, and predictive approaches seems therefore a proper
solution to estimate environmental concentrations. The re-
liability of predictive approaches for studying environmen-
tal contamination by pharmaceuticals was recently tested
by comparing predicted and measured concentrations in
environmental media (Calamari et al. 2003, Castiglioni et
al. 2004b). Two different approaches, a mass balance calcu-
lation, and an equation proposed by the European Agency
for the Evaluation of Medicinal Products (EMEA), were used
to calculate the predicted environmental concentrations
(PECs) of a group of top pharmaceuticals. PEC were then
compared with the measured environmental concentrations
(MECs) in the rivers Po and Lambro in Italy. At the begin-
ning, PECs were calculated by a mass balance approach,
dividing the theoretical loads by the average annual flow
rate of the rivers at each sampling site.
The theoretical environmental loads were in turn estimated
by normalizing the nation-wide sales figures to the number
of inhabitants of the drainage basin at the sampling stations.
In a second study, 'crude' PECs were also calculated using
the equation suggested by the EMEA, that took into account
the predicted amount of pharmaceuticals used per year in
the test area, the number of inhabitants, the volume of waste
water per capita per day, and a dilution factor for waste
water in surface water. These PEC values were then refined
by correcting the 'crude' data for the metabolic rates, i.e.
the percentage of the pharmaceuticals excreted as the par-
Po River
Maximum
37.0
67.2
41.7
24.4
34.2
4
26.2
Nd
17.4
43.8
2.7
15.9
248.9
20.3
1.7
Nd
Nd
Nd
0.1
Nd
Nd
Nd
Median
33.1
3.5
17.2
4.6
23.1
1.3
Nd
Nd
13.0
9.8
1.9
3.2
32.6
1.6
1.1
Nd
Nd
Nd
0.1
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd
Nd

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ent compound by humans (obtaining the PECm) and the
rate of degradation of the pharmaceuticals in the environ-
ment (obtaining the PECe). Crude PEC values for the River
Po calculated by the mass balance approach were two to
three times lower than those calculated using the EMEA
method, and in general provided a better approximation of
the MEC. Refined PEC, calculated using the EMEA method,
provided a good approximation of the MEC in about 40%
of the cases, while PEC and MEC differed by one order of
magnitude in 30% and two orders of magnitude in the other
30% (Castiglioni et al. 2004b). This was a first attempt to
compare predicted and measured concentrations and the
MEC/PEC ratio was useful to test the consistency of the
findings, to identify outliers and anomalous situations, and
to assess methods for calculating PECs, for testing the reli-
ability of predictive approaches for pharmaceuticals in the
environment (Calamari et al. 2003).
6
The widespread occurrence of pharmaceuticals in aquatic
habitats has raised a novel and intricate environmental is-
sue. The potential risk associated with the presence of these
contaminants in surface waters is mostly unknown. The ef-
fects of active pharmaceutical principles on aquatic life and/
or humans, influencing water for drinking and other health
related activities, have scarcely been investigated. Toxicity stud-
ies in this field have to face a number of difficulties. Drugs are
present in the environment at very low concentrations, far
below the doses employed for medical treatments, so acute
toxicity tests do not detect any biological effects (Boxall et
al. 2003). Target and non-target organisms are therefore
exposed to low levels of pharmaceuticals in the environ-
ment, but for very long periods of time. Therapeutic drugs
are ordinarily found in complex mixtures of unrelated chemi-
cals with a range of pharmacological activities, suggesting
additive and synergic effects (Cleuvers 2003). This raises
questions about the possible danger posed by water-borne
drugs on the survival and physiology of aquatic life, or their
ability to induce genetic alterations or mutations.
Investigating how pharmaceutical pollutants might act on
aquatic photosynthetic organisms, a recent paper reported
the effects of erythromycin, tetracycline and ibuprofen on
the growth of the cyanobacterium Synechocystis sp. and the
duckweed Lemna minor (Pomati et al. 2004a). The three
compounds were chosen as they are widely present in sev-
eral types of European surface waters and sediments, with
concentrations up to µg L–1 (Hirsh et al. 1999, Zuccato et
al. 2000). Significant differences were observed in their ef-
fects on Synechocystis and Lemna. While erythromycin af-
fected the growth of both organisms, tetracycline had a slight
inhibitory effect on Synechocystis and promoted growth in
Lemna at the level of 10 µg L–1. The anti-inflammatory
ibuprofen strongly stimulated the proliferation of Synecho-
cystis at all concentrations tested, but inhibited Lemna
growth in a linear dose-dependent manner. Erythromycin
inhibitory effect on Lemna was unexpected, and this eu-
karyotic organism was the most sensitive ever tested against
ibuprofen to date (Pomati et al. 2004a). Exposure to the
three drugs also resulted Lemna producing the stress hor-
mone abscisic acid (ABA) (Pomati et al. 2004a). These re-
Ecotoxicology
sults indicated a possible physiologic impact of water-borne
drugs on aquatic plants, similar to general oxidative stress.
At present we are investigating the toxicity of 13 therapeu-
tic principles, combined in a mixture that mimics the con-
centration (ng/L) and combinations of drugs detected in north-
ern Italian rivers (Calamari et al. 2003, Castiglioni et al. 2004b).
The mixture comprises atenolol, bezafibrate, carbamazepine,
cyclophosphamide, ciprofloxacin, furosemide, hydrochlorothi-
azide, ibuprofen, lincomycin, ofloxacin, ranitidine, salbutamol,
and sulphamethoxazole. We employed in vitro toxicological
tests using cell clones, to minimise the variability in biological
response in different individuals. We chose zebrafish liver cells
(ZFL) and human embryonic cells (HEK293) as the cellular
models representative of a target aquatic animal and the most
sensitive human archetype, respectively (Ghosh et al. 1994,
Inoki et al. 2003). The specific objectives were to evaluate the
combined effects of drugs on cells growth and to elucidate the
mechanisms of toxicity at the molecular level by studying the
expression of genes and proteins. Preliminary results indicated
that, at environmental concentrations, therapeutic drugs in-
hibited the growth of both ZFL and HEK293 cells in vitro,
with the highest effects for 72 h exposure, corresponding to a
30% decrease in proliferation compared to controls (Pomati
et al. 2004b). In HEK293 cells, concomitant with reduced
proliferation, we noted activation of functional pathways
linked to cellular stress responses such as extracellular signal-
regulated kinases, and with the control of cell cycle progres-
sion. Results were similar on DNA microarray-based analysis
of gene expression in ZFL cells exposed to environmental lev-
els of the drug mixture (Pomati et al., unpublished). These
results suggest that a combination of pharmaceuticals at envi-
ronmental levels (ng/L) can significantly inhibit cell growth in
vitro, and that cellular specialization/differentiation could be
a target process affected by contamination by therapeutic drugs
in the environment.
Another potential effect for aquatic life and/or humans is
related to the presence of antibiotics in waste, surface and
ground waters. Exposure to antibiotics might induce anti-
microbial resistance (Kummerer 2004b), and horizontal
transfer of resistance genes in field bacterial populations
(Davidson 1999). Correlations between antibiotics in the
environment and the development of resistance in bacteria
were recently investigated using the multidrug-resistance gene
marA. marA is located in the marRAB operon that is in-
duced by tetracycline, chloramphenicol, ampicillin, nalid-
ixic acid and ciprofloxacin, and its expression is sufficient
to produce a multidrug resistance phenotype (Goldman et
al. 1996). Samples of sediment and water were collected
from rivers and treatment plants and genomic DNA was
extracted. Samples were plated with antibiotics using the
disk diffusion technique. PCR found marA in almost all
samples, and all the plated samples gave rise to bacterial
colonies belonging to the genus Bacillus and resistant to at
least three of the five antibiotics tested. PCR confirmed the
presence of marA in the plated colonies. These preliminary
results suggest that the gene marA is not directly related to
the presence of antibiotics, but is widespread in environ-
mental samples (Castiglioni et al. 2005b), thus indicating
the environment as a reservoir of resistance (Biyela et al.
2004). The persistence of bacteria resistant to antibiotics,

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Pharmaceuticals in the Environment in ItalyReview Articles
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ESPR – Environ Sci & Pollut Res 13
13
13 13
13 (1) 2006
despite a decrease in antibiotic prescriptions, have been re-
ported for sulphonamide in UK (Enne et al. 2001), but the
ban of antibiotics as growth promoters in animal farming,
such as for vancomycin in Sweden (1986) and for avoparcin
in Denmark (1995) (Van de Bogaard & Stobberingh 1999),
resulted in a significant decrease of resistance in manure and
enteric bacteria. Recently, Cunha (Cunha 2001) suggested
that the development of antibiotic resistance might not be
related to the total amounts of antibiotics used, but to the
potency of the single compounds.
7
Hundreds of tons of pharmacologically active substances can
enter sewage treatment plants (STPs) each year. For the ma-
jority of drugs, removal by conventional biological treatments
seems inefficient (Ternes 1998, Stumpf et al. 1999, Metcalfe et
al. 2003, Carballa et al. 2004), since they are found in signifi-
cant amounts in STP effluents and surface water (Hirsch et al.
1999, Calamari et al. 2003). STPs might therefore be impor-
tant point sources of contamination, but for the majority of
pharmaceuticals little information is available on their
behaviour and ultimate fate in STPs and in the receiving sur-
face water. The removal rate of pharmaceuticals in STPs is
potentially affected by several factors, such as the nature of the
drug, the treatment process employed, the age of the activated
sludge, environmental conditions such as the season, and the
characteristics of the influent (O'Brien & Dietrich 2004).
A recent study used mass balance calculation to investigate
six STPs in Italy to gain further knowledge of the removal
and behaviour of drugs during treatment. Removal efficien-
cies were assessed for 30 compounds and the factors affect-
ing removal in STPs were identified (Castiglioni et al. 2004a).
Some STPs (in Naples, Latina and Cuneo) were sampled in
winter (January–March), others (Cagliari and Varese Lago)
in summer (June–September); only Varese Olona was
sampled in both seasons. Removal rates were calculated by
comparing the load of each drug in influents and effluents
at each plant. Considering the total loads, removal rates were
generally less than 40%, with the exception of the Varese
Lago plant, were it was 64%. Removal rates were 0, 16, 31
and 39% in the four STPs sampled in winter and 0, 31 and
64% in those sampled in summer. Where a comparison was
possible (Varese Olona), removal rates seem to be higher in
summer than winter (31% versus 0%), in line with a tem-
perature-dependent increase of microbial activity. Seasonal
differences were also observed for the removal rates of the
single substances. Pharmaceuticals could be grossly divided
into three groups. A first group had removal rates higher in
summer than in winter, a second group had similar removal
rates in summer and winter, and the last group had re-
moval close to zero in both seasons. Total loads in summer
were about half those in winter (1.5 versus 3 g/day/1000
inhabitants in the Varese Olona STP) probably because phar-
maceuticals, particularly antibiotics, are used less in sum-
mer. For the antibiotics ciprofloxacin, ofloxacin and sulpha-
methoxazole the loads were significantly lower in summer
than winter, while for other pharmaceuticals, such as aten-
olol, furosemide, ibuprofen, hydrochlorothiazide and raniti-
dine, the loads were similar in both season.
STPs, Removal Efficiency and Mitigation
This study therefore found that the removal rates of phar-
maceuticals in STPs can vary significantly over the year, but
abatement is always incomplete. Tertiary treatments such
as flocculation, ozonation, advanced oxidation, membrane
filtration (activated carbon adsorption), and photocatalysis
could be used to improve removal in the near future. Since
preventing the drugs entering the aquatic ecosystem might
be a key strategy for reducing the environmental contami-
nation, the possibilities offered by these new technologies
need proper evaluation.
8
Pharmaceuticals in the environment are increasingly a matter
of global concern, with potential consequences for humans
and the environment. However, the effects of life-long expo-
sures have still to be determined. For instance, we do not know
whether this spread of antibiotics in the environment contrib-
utes to bacterial resistance, or to what extent drugs can be
transferred to humans through food-chain biomagnification.
Research needs to be encouraged and the human and environ-
mental implications of the massive discharge of pharmaceuti-
cals into the environment calls for thorough evaluation. Sev-
eral thousand tons of medicinally active substances are used
every year in the world for humans and animals, and excreted
as parent compound or active metabolites, escaping degrada-
tion in STPs, and entering fresh water. Until recently, informa-
tion on medicinal substances released into the environment
was scant but there are now numerous studies measuring them
in effluents of STPs and surface and ground water world-wide.
Knowledge is rapidly growing, but nevertheless the problem
is not yet fully clear, and information on risk assessment and
management is far from adequate. We still have to boost our
knowledge of the causes, occurrence and effects of drugs as
environmental pollutants for a better understanding of this
recent ecological issue, to improve abatement strategies, and
to mitigate subtle environmental consequences.
Conclusions
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Received: July 21st, 2005
Accepted: October 9th, 2005
OnlineFirst: October 10th, 2005