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Journal of Hazardous Materials 179 (2010) 1042–1048

Contents lists available at ScienceDirect

Journal of Hazardous Materials

journa l homepage: www.e lsev ier .com/ locate / jhazmat

ource, occurrence and fate of antibiotics in the Italian aquaticnvironment

ttore Zuccato ∗, Sara Castiglioni, Renzo Bagnati, Manuela Melis, Roberto Fanelliepartment of Environmental Health Sciences, Mario Negri Institute for Pharmacological Research, Via La Masa 19, 20156 Milan, Italy

r t i c l e i n f o

rticle history:eceived 9 December 2009eceived in revised form 26 March 2010ccepted 26 March 2010vailable online 1 April 2010

a b s t r a c t

Aim of this study was to provide an up-to-date assessment of the antibiotics contaminating the aqueousenvironment in Italy, for a better understanding of risks for the ecosystem and human health. Antibioticswere first listed in order of their theoretical environmental loads, then were measured in wastewater ofsome sewage treatment plants (STPs) and in rivers in Italy. Macrolides, particularly clarithromycin andspiramycin, and quinolones, particularly ciprofloxacin and l-floxacin/ofloxacin, were the most abundant

eywords:ntibioticsiquid chromatography–tandem masspectrometry (HPLC–MS–MS)

aste waterewage treatment plant (STP)

antibiotics in untreated wastewater. Several of them were not removed in STPs and still remained inthe treated wastewater, and a total estimate of 7–14 tons of active principles were discharged annuallyinto the aqueous environment in Italy through this route. Results of the analysis of rivers in northernItaly agreed with these figures, with an average load of 5 kg/day, or about 1.8 tons/year, of antibioticsflowing in the River Po, at sampling sites covering a basin comprising about one-fifth of the Italian popu-lation. In conclusion, antibiotics, particularly macrolides and quinolones, are widespread environmental

STPs

urface water contaminants, and urban

. Introduction

Antibiotics are widely used to treat or prevent infective diseasesn human and veterinary medicine. Large amounts are also used ingriculture to promote fruit growth, in bee-keeping and in livestocknd fish farming as growth promoters [1,2]. The global market con-umption of antibiotics was recently estimated at between 100,000nd 200,000 tons/year [3]. Most of these compounds are not com-letely metabolized and residues of the antibiotics used in humansre excreted with urine and feces, reaching urban sewage treat-ent plants (STPs), where they may escape degradation and can

ontaminate waste, surface and ground-water [4–8]. STPs are con-idered major contributors to the spread of human antibiotics in thenvironment. Antibiotics given to livestock can also be dispersedn fields through manure and can reach soil and ground-water,

hile those used in crops and fish farming can accumulate in soilr sediment, thus contributing to the contamination [9,10].

This load of antibiotics dispersed in the environment couldave important consequences for ecosystems and human health,

ossibly contributing to the increase of allergies in humans andhe spread of antibiotic-resistant bacteria. Continuous exposure tontibiotics can enhance the selection of resistant bacterial strains inhe environment [11]. Bacterial resistance to antibiotics has been

∗ Corresponding author. Tel.: +39 02 39014544; fax: +39 02 39014735.E-mail address: zuccato@marionegri.it (E. Zuccato).

304-3894/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.jhazmat.2010.03.110

are confirmed a major source of the contamination.© 2010 Elsevier B.V. All rights reserved.

reported in sewage [12], surface water [13] drinking water [14],farm soil [15] and marine aquaculture sites [16]. Antibiotics aretypically found in the environment at sub-therapeutic concentra-tions, which can in fact promote bacterial resistance [17]. Aquaticand soil ecosystems seem to act as reservoirs of antibiotic-resistantbacteria [18,19], although the implications for human health arestill unknown. All this raises questions for human health and thestability of the ecosystem.

This paper presents and discusses results from some recent ana-lytical campaigns, to assess the presence and concentrations of themain antibiotics for human use consumed in Italy, in untreated andtreated wastewater of some STPs in northern Italy and in receiv-ing rivers. The aim is to provide a qualitative and quantitativeassessment of the antibiotics entering the environment, for a betterunderstanding of the risk for the ecosystem and human health.

2. Materials and methods

2.1. Identification of the antibiotics accounting for the bulk ofpollution

Environmental contamination by pharmaceuticals needs to be

monitored for several reasons, including reliable assessment ofrisks for the environment and for man. However, thousands of dif-ferent pharmaceuticals are used regularly so blanket monitoringis unthinkable because of the excessive number of drugs with dif-ferent chemical structures and physico-chemical properties. It is

E. Zuccato et al. / Journal of Hazardous Materials 179 (2010) 1042–1048 1043

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ig. 1. Map of the locations sampled in Italy and Switzerland. Waste-water was cohe River Po (S1–S4) and the River Arno (S1–S4). S = sampling site.

herefore best to focus on molecules of concern for the environ-ent and to establish priorities so as to restrict studies to a limited

umber of important hazardous molecules.In previous studies in Italy we established a list of priority phar-

aceuticals for human use and identified potential contaminants,ased on their mass balance and theoretical loads, by multiplyingales by the percentages of metabolic excretion in humans [6,9].his provided a list of ‘top’ pharmaceuticals for human use, excretednmetabolized in substantial amounts, with estimated environ-ental loads in the order of tons per year [8]. This also applies

o antibiotics which we submitted to a preliminary selection, todentify a restricted list of those most likely to cause environmentalroblems. A second group of antibiotics, used in Italy as growth pro-oters (tylosin, tilmicosin, oleandomycin, oxytetracycline), was

ubsequently added to the list to check environmental contamina-ion from veterinary medicines. The complete list of the antibioticselected for this investigation with the respective therapeutic cat-gories is reported in Tables 1–4.

.2. Sampling sites and sample collection

.2.1. Sewage treatment plantsIn various periods of the year in 2007 and 2008 we sampled

our STPs in Milan, Varese and Como (Italy) and Lugano (Switzer-and) (Fig. 1). For each plant, composite 24 h samples of influentsnd effluents were obtained by pooling wastewater collected every0 min by an automatic sampling device. Effluents were sampledome time after influents to take into account the residence timef the wastewater in the plants. Water samples (500 mL) wererozen and stored at −20 ◦C until analysis. In the Varese STP threeludge samples were also collected from the activated sludge tanks.ludge was immediately lyophilized and stored at−20 ◦C until anal-sis.

One STP was in a large city (Milan), and three smaller ones ineighboring towns (Varese and Como in Lombardy and Lugano

n Switzerland). The STPs were all equipped with pre-treatment,rimary and secondary treatment facilities, i.e. primary settlingnd activated sludge processes, and receive mainly domestic

d in four STPs in Milan, Varese, Lugano and Como, river water was collected along

waste. The Varese STP also had UV-light tertiary treatment. Allthe plants discharged the treated water directly into rivers orlakes.

The Milan STP serves a population equivalent of 1,250,000inhabitants, has a mean flow rate of 374,000 m3/d and dischargesthe treated water into irrigation canals that flow into the River Lam-bro and finally into the River Po. The Varese STP has a populationequivalent of 229,000, a flow rate of 94,000 m3/d and discharges thetreated water into irrigation canals that flow into the River Ticinoand then into the Po. The Como and Lugano STP have respectively101,000 and 120,000 inhabitants, and 63,000 and 61,000 m3/dflow rates. Their treated waters are discharged into canals flowingrespectively into the Como and Lugano lakes.

2.2.2. Rivers Po and ArnoComposite samples were obtained by pooling river water sam-

ples collected every 20 min for 2 h by a portable automatic sampler(Sigma 900 Standard, Hach Company, USA). River sampling siteswere chosen with the aim of collecting surface waters downstreamof largely populated areas in northern (River Po) and central Italy(River Arno) (Fig. 1).

The River Po, the largest Italian river, was sampled down-stream of the inlets of the main influents, and downstream ofthe major towns. The first sampling site was at Mezzana Corti(225 km from the source, flow rate 606 m3/s) upstream of the con-fluence with the River Ticino. The other sampling sites were atMonticelli Pavese (310 km from the source, flow rate 743 m3/s),after the confluence of the River Ticino, Piacenza (337 km fromthe source, flow rate 874 m3/s) and Cremona (386 km from thesource, flow rate 984 m3/s) after the confluence of the River Lam-bro.

The River Arno was sampled at Rignano sull’Arno (102 km fromthe source, flow rate 4 m3/s) and Limite sull’Arno (156 km from the

source, flow rate 9.9 m3/s), respectively upstream and downstreamof Florence. Other sampling sites were at Castelfranco (180 km fromthe source, flow rate 10 m3/s) and near the city of Pisa (223 km fromthe source, flow rate 9.7 m3/s), upstream and downstream of somemajor tributaries and the Pisa STP.

1044 E. Zuccato et al. / Journal of Hazardous Materials 179 (2010) 1042–1048

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ig. 2. Sample and standard HPLC-MRM chromatograms of the most abundant1 ng/injected).

.3. Analysis

.3.1. Chemicals and materialsThe list of the antibiotics selected for analysis includes sev-

ral antibacterial drugs: penicillins, quinolones, macrolides,incosamides, glicopeptides, sulphamides and tetracyclines. Theeference standards of amoxicillin, erythromycin, lincomycin,-floxacin/ofloxacin, oxytetracycline, spiramycin, clarithromycin,leandomycin, tylosin, tilmicosin and sulphamethoxazoleere purchased from Sigma–Aldrich (Steinheim, Germany);

iprofloxacin was purchased from ICN Biochemicals (Meckenheim,ermany). Dehydro-erythromycin was synthesized according tocArdell et al. [20]. Briefly, erythromycin-H2O was obtained by

djusting the pH of an erythromycin solution to 3.0 and stirringt for 4 h at room temperature. The compounds used as internaltandards (I.S.), salbutamol-D3 (99.1% D) and ibuprofen-D3 (99.7%), were purchased from CDN Isotopes (Quebec, Canada). All the

eference standards were stored at 4 ◦C.Standards were dissolved in methanol to a concentration of

mg/mL and subsequently diluted to 10 ng/�L (stock solution) and–0.1 ng/�L (working solutions). The stock solutions of amoxicillin,iprofloxacin, ofloxacin, oxytetracycline and sulphamethoxazoleere renewed monthly because of their limited stability. The I.S.ere dissolved in methanol (1 mg/mL) and subsequently diluted

iotics. Left column: untreated waste-water. Right column: analytical standards

to 10 and 1 ng/�L. All stock and I.S. solutions were stored at −20 ◦Cin the dark.

The cartridges used for solid phase extraction were: 3 mL dis-posable OASIS MCX (60 mg, Waters Corp., Milford, MA) and 3 mLdisposable Lichrolut EN (200 mg, Merck, Darmstadt, Germany). Allthe solvents were of reagent grade or higher. Acetone, methanol,ethyl acetate were for pesticide analysis (Carlo Erba Reagents,Italy), acetonitrile for LC–MS (Riedel de Haen, Seelze, Germany).Ammonium hydroxide solution (25%), formic acid (98–100%) andtriethylamine (99.5%) were from Fluka (Steinheim, Germany).Hydrochloric acid (37%) was from Carlo Erba (Milan, Italy). HPLCgrade Milli-Q water was obtained with a MILLI-RO PLUS 90 appa-ratus (MILLIPORE, Molshelm, France).

2.3.2. Solid phase extractionWaste and surface water samples were extracted as described

in previous publications [8,21,22], where detailed informationon recoveries and performance of the methods are reported.Briefly, water samples were filtered on glass microfiber filters

GF/A 1.6 �m (Whatman, Kent, UK) and spiked with internal stan-dards before extraction. The extraction was done on two SPEcolumns, an Oasis MCX at pH 2.0 for amoxicillin, ciprofloxacin,l-floxacin/ofloxacin, lincomycin, sulphamethoxazole, vancomycin,oleandomycin, oxytetracycline and tilmicosin, and a Lichrolut EN

E. Zuccato et al. / Journal of Hazardous Materials 179 (2010) 1042–1048 1045

Table 1Loads (mg/day/1000 inhabitants) of antibiotics in influents and effluents of the four STPs.

Antibiotics Milan (mean ± SD) 7days monitoring

Varese (mean ± SD) 3days monitoring

Lugano (mean ± SD) 7days monitoring

Como (mean ± SD) 7days monitoring

Influent Effluent Influent Effluent Influent Effluent Influent Effluent

PenicillinsAmoxicillin nd nd 6.05 ± 10.5 nd 382 ± 453 37 ± 24 21 ± 15 nd

QuinolonesCiprofloxacin 32 ± 12 24 ± 4.5 203 ± 121 60 ± 46 4.9 ± 6.0 Nd 108 ± 15 ndl-floxacin/ofloxacin 7.7 ± 6.9 5.3 ± 1.6 184 ± 134 77 ± 56 0.31 ± 0.60 nd 20 ± 25 4.9 ± 8.4

Macrolides–lincosamidesClarithromycin 322 ± 74 104 ± 28 121 ± 60 52 ± 41 318 ± 45 437 ± 70 939 ± 407 500 ± 80Erythromycin 0.39 ± 0.3 34 ± 5.3 4.6 ± 2.8 27 ± 15 0.56 ± 1.1 59 ± 12 nd 6.5 ± 1.2Erythromycin-H2O na na na na na na 25 ± 5.7 17 ± 4.5Lincomycin 15 ± 3.7 4.9 ± 1.5 3.9 ± 3.0 2.8 ± 1.5 0.54 ± 0.2 1.1 ± 1.2 10 ± 6.6 5.8 ± 2.7Spiramycin 24 ± 11 121 ± 30 234 ± 155 146 ± 63 1.7 ± 1.5 393 ± 179 97 ± 23 75 ± 24

SulphamidesSulfamethoxazole 40 ± 21 16 ± 2.8 97 ± 38 11 ± 19 33 ± 15 15 ± 11 33 ± 44 30 ± 5.6

GlycopeptidesVancomycin 9.6 ± 1.8 5.2 ± 0.5 14 ± 20 10 ± 13 nd 24 ± 13 nd 9.4 ± 6.7

Veterinary antibioticsOleandomycin nd nd 0.31 ± 0.50 1.2 ± 0.3 nd nd nd ndOxytetracycline nd 0.24 ± 0.50 nd nd nd nd nd ndTilmicosin nd nd nd nd nd nd nd ndTylosin 4.0 ± 3.0 1.6 ± 1.7 nd nd nd nd nd nd

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Total loads (mg/day/1000 inhabitants) 459 ± 76 317 ± 58 869 ±a = not assessed; nd = not detectable (concentrations < LOQ).

t pH 7.0 for clarithromycin, erythromycin, erythromycin-H2O,piramycin and tylosin. Wastewater and river water samples,espectively 50 and 500 mL aliquots, were spiked with 20 ng ofnternal standards (salbutamol-D3 and ibuprofen-D3). The Oasis

CX cartridges were conditioned before use with 6 mL methanol,mL Milli-Q water and 3 mL water acidified to pH 2. Samplesere then passed through the cartridges under vacuum, and the

artridges were vacuum-dried for 5 min. Elution was with 3 mLethanol, and 3 mL 2% ammonia solution in methanol. The Lichro-

ut EN cartridges were conditioned before use with 6 mL methanolnd 6 mL Milli-Q water. Cartridges were vacuum-dried for 10 minnd eluted with 3 mL methanol and 3 mL ethyl acetate. All the elu-tes were pooled and dried under a nitrogen stream.

An ultrasonic solvent extraction (USE) was used for sludge sam-

les extraction as previously described for particulate [22]. Sludge5 g) was extracted three times with 20 mL methanol, and afterach extraction step, the samples were centrifuged for 10 min at000 rpm. The supernatants were finally pooled, dried under an

ig. 3. Total amounts of antibiotics discharged into the environment (g/1000 inhab-tants/year) in the cities investigated, and removal rates in the STPs.

388 ± 69 740 ± 438 966 ± 241 1261 ± 543 653 ± 135

air stream, redissolved in 100 �L Milli-Q water and filtered beforeanalysis.

The instrumental limits of quantification (IQL) were in the hun-dreds pg/injected range, and the limits of quantification (LOQ) ofthe method were in the low ng/L range (0.5–2 ng/L) [8,21,22].

2.3.3. HPLC–MS–MS analysisSamples were analysed by reversed-phase liquid

chromatography–tandem mass spectrometry (HPLC–MS–MS).Samples were analysed with an HPLC system consisting of twoPerkin-Elmer Series 200 pumps and a Perkin-Elmer Series 200auto sampler, and an API 3000 triple quadrupole (Q1q2Q3) massspectrometer equipped with a turbo ion spray source (AppliedBiosystems-Sciex, Thornhill, Ontario, Canada). A Luna C8 column50 mm × 2 mm i.d., 3 �m particle size (Phenomenex, Torrance, CA,USA) was used for chromatographic separation at a flow rate of200 �L/min. Mass spectrometric analysis was done in the multiplereaction monitoring mode (MRM), in negative (sulphamethoxa-zole) and positive ionisation modes (all the other compounds).Quantification was by isotope dilution using ibuprofen-D3 forsulphamethoxazole and salbutamol-D3 for the other antibiotics.

The chromatographic and mass spectrometric conditions arereported in detail in previous papers [8,21,22]. Some examplesof sample (untreated wastewater) and standard (1 ng/injected)chromatograms are reported in Fig. 2 for the most abundant antibi-otics.

3. Results and discussion

3.1. Antibiotics in wastewater

Table 1 shows the loads of the antibiotics in influents and efflu-

ents of the Milan, Como, Lugano and Varese STPs expressed asmg/day/1000 inhabitants. Macrolides, particularly clarithromycinand spiramycin in all the locations, and quinolones (ciprofloxacinand l-floxacin/ofloxacin) in Varese and Como, were the most abun-dant antibiotics in wastewater. Results were similar for macrolides,

1046 E. Zuccato et al. / Journal of Hazardous Materials 179 (2010) 1042–1048

Table 2Behaviour and fate of antibiotics in the Varese STP.

Antibiotics Concentration (ng/L) Concentration (ng/kg)

Influent Effluent pre-UV (% removala) Effluent post-UV (% removalb) Sludge

PenicillinsAmoxicillin 18 nd (100) nd nd

QuinolonesCiprofloxacin 513 147 (71) 148 (0) 2090l-floxacin/ofloxacin 463 235 (49) 191 (19) 3408

Macrolides–lincosamidesClarithromycin 319 117 (63) 145 (0) 156Erythromycin 12 52 (0) 72 (0) 185Erythromycin-H2O na na na naLincomycin 9.7 6.1 (37) 7.2 (0) ndSpiramycin 603 454 (25) 375 (17) 658

SulphamidesSulfamethoxazole 246 46 (81) 101 (0) nd

GlycopeptidesVancomycin 41 40 (2) 29 (28) nd

Veterinary antibioticsOleandomycin 2.2 2.4 (0) 3.1 (0) ndOxytetracycline nd nd nd ndTilmicosin nd nd nd ndTylosin nd nd nd nd

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a = not assessed; nd = not detectable (concentrations < LOQ).a Difference between the influent and the effluent pre-UV.b Difference between the effluents pre-UV and post-UV.

uinolones and sulphamides in the effluents from eight STPs inanada [23] and in the influents and effluents of several STPs inI, USA [24].Loads in treated wastewater were generally lower than in

ntreated water, with some exceptions. In some cases spiramycinnd erythromycin were more abundant in effluents than in

nfluents, because they can bind to sludge and are releasedater (see below). Similar results were obtained for spiramycin,rythromycin-H2O and clarithromycin in two municipal STPsn Switzerland [25] and for erythromycin in the UK [26]. Sul-hamethoxazole was also found in significant amounts in influents

able 3ntibiotic concentrations (ng/L) in the River Po.

Antibiotics River Po

Mezzana corti Monticelli PV

PenicillinsAmoxicillin <2.08 <2.08

QuinolonesCiprofloxacin 2.24 1.32l-floxacin/ofloxacin 11.48 0.65

Macrolides–lincosamidesClarithromycin 1.78 0.89Erythromycin 0.78 3.51Erythromycin-H2O 1.66 4.27Lincomycin 5.46 3.72Spiramycin <1.4 <1.4

SulphamidesSulfamethoxazole 1.83 2.39

GlycopeptidesVancomycin 0.59 4.88

Veterinary antibioticsOleandomycin <0.31 <0.31Oxytetracycline <1.19 <1.19Tilmicosin <0.7 <0.7Tylosin <0.77 <0.77

a Values below the LOQ were taken as half the LOQ.

and effluents, while loads of the other antibiotics for human usewere generally lower and variable, particularly amoxicillin, andveterinary antibiotics were present only in traces.

Fig. 3 shows an estimate of the total load of antibiotics still per-sisting in the treated wastewater of the STPs investigated. Removalrates (RRs) in STPs varied but generally did not exceed 50%. As a con-

sequence an average amount of 115–237 g of antibiotics per 1000inhabitants/year (in Milan, Como and Varese) and about 350 g/1000inhabitants/year in Lugano, still persisted in the treated wastewa-ter and could therefore be released into the receiving water anddischarged into the environment.

Mean ± SD

Piacenza Cremona

<2.08 <2.08 <2.08

16.00 15.50 8.8 ± 8.118.06 13.30 10.9 ± 7.4

2.19 1.89 1.7 ± 0.64.62 2.82 2.9 ± 1.65.31 3.63 3.7 ± 1.56.22 7.47 5.7 ± 1.62.35 <1.4 1.1 ± 0.8a

2.26 2.11 2.1 ± 0.2

11.69 2.20 4.8 ± 4.9

<0.31 <0.31 <0.311.82 1.23 1.1 ± 0.6a

8.93 <0.7 2.5 ± 4.3a

<0.77 <0.77 <0.77

E. Zuccato et al. / Journal of Hazardous Materials 179 (2010) 1042–1048 1047

Table 4Antibiotic concentrations (ng/L) in the River Arno.

River Arno

Antibiotics Rignano sull’Arno Limite sull’Arno Castelfranco Pisa Mean ± SD

PenicillinsAmoxicillin 3.57 3.77 5.57 9.91 5.7 ± 2.9

QuinolonesCiprofloxacin 10.55 <1.8 26.89 37.50 19 ± 16.4a

l-floxacin/ofloxacin 1.68 <1.4 10.88 6.70 5 ± 4.7a

Macrolides–incosamidesClarithromycin 6.70 16.55 33.59 44.76 25.4 ± 17Erythromycin 3.91 2.88 6.81 8.12 5.4 ± 2.4Erythromycin-H2O 13.96 9.68 17.29 30.52 17.9 ± 9Lincomycin 8.72 5.34 10.92 7.41 8.1 ± 2.3Spiramycin <1.4 6.79 17.92 6.19 7.9 ± 7.2a

SulphamidesSulfamethoxazole 1.79 11.40 3.93 3.97 5.3 ± 4.2

GlycopeptidesVancomycin 0.44 1.10 3.58 5.17 2.6 ± 2.2

Veterinary antibioticsOleandomycin <0.31 <0.31 <0.31 <0.31 <0.31Oxytetracycline <1.19 <1.19 <1.19 <1.19 <1.19

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Aggregate results of the campaigns on the Rivers Po and Arno arereported in Fig. 4. The River Po is the largest Italian river, with a flowrate of 606–984 m3/s (about 50–80 million m3/day) at the samplingsites. The total loads of antibiotics carried by the river were about

Tilmicosin 1.97 <0.71Tylosin <0.77 <0.77

a Values below the LOQ were taken as half the LOQ.

.2. Behaviour and fate of antibiotics in a STP

The behaviour and fate of the antibiotics analysed were inves-igated in the Varese STP where, after primary mechanical andecondary biological treatments, wastewater undergoes a ter-iary treatment with UV-light. Table 2 shows the concentrationsf the antibiotics in the STP influent and effluent before andfter UV-light treatment, and in samples of sludge, collectedoncomitantly with the sampling of the influent. This tableonfirms that macrolides, particularly clarithromycin and spi-amycin, quinolones (ciprofloxacin and l-floxacin/ofloxacin) andulphamethoxazole were the most abundant antibiotics in wastew-ter. RRs were particularly high for amoxicillin (100%), >50% foruinolones, sulphamethoxazole and clarithromycin, 25–50% for

incomycin and spiramycin, and <5% for erythromycin, vancomycinnd the veterinary antibiotic oleandomycin. UV-light had limitedffect on RR, except for l-floxacin/ofloxacin (19%), spiramycin (17%)nd vancomycin (28%).

Significant concentrations of the quinolones (l-oxacin/ofloxacin and ciprofloxacin) and lower concentrations ofhe macrolides spiramycin, erythromycin and clarithromycin wereound in sludge. Quinolones were also detected in sewage sludgeamples from different STPs and in sludge-treated soil samples inwitzerland, confirming their ability to bind to sludge and soils9].

.3. Antibiotics in surface water

Tables 3 and 4 report concentrations of the antibiotics in theivers Po and Arno. The concentration generally increased fromource to mouth in both rivers, due to the loads of antibioticsntering surface water with the treated urban discharges thattill contain large amounts of these substances. The most abun-ant compounds in the River Po (Table 3) were ciprofloxacin,

floxacin, lincomycin and vancomycin with mean concentrationsetween 5 and 10 ng/L. The other compounds were in the highg/L range, or undetectable. In the River Arno (Table 4) theost abundant compounds were ciprofloxacin, clarithromycin and

rythromycin-H2O (mean concentration about 20 ng/L). All the

6.67 <0.71 2.3 ± 3.0<0.77 <0.77 <0.77

other compounds were in the low ng/L range, and veterinaryantibiotics were mostly undetectable. During a recent analyti-cal campaign in the River Seine ciprofloxacin and ofloxacin weredetected at levels comparable with those found in our study in Italy,while the concentration of sulphamethoxazole was at higher level[27].

Fig. 4. Antibiotic loads along the course of the Rivers Po and Lambro. S = samplingsite (see Fig. 1).

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.2 kg of active ingredients per day in the first two sampling sites,ising to about 5.8 and 4.2 kg/day at the third and fourth samplingites respectively (Fig. 4).

In the Arno, a major river in central Italy, with a flow rate of about0 m3/s (860,000 m3/day), total loads substantially increased fromource to mouth, up-to about 130 g per day.

. Conclusions

Antibiotics are widely used in human and veterinary medicinend most of them can also be considered widespread environ-ental contaminants. However, in spite of being extensively

nvestigated, systematic data on antibiotics are scarce and insuffi-ient for an environmental risk assessment [28]. Several scatteredurveys have been carried out throughout Europe, but despite theumerous studies performed there is still a lack of understand-

ng and knowledge. In particular there is a substantial lack ofystematic studies addressing this issue. This paper deals with aystematic study on antibiotics occurrence and fate in the environ-ent. Antibiotic prescriptions have been studied systematically in

taly, a priority list was obtained on the basis of prescriptions andhe listed substances were measured in the environment, obtain-ng evidences of the occurrence of some of these molecules, butot of others. Analysis of a group of antibiotics, including theost frequently prescribed penicillins, quinolones, macrolides, lin-

osamides, sulphamides and glycopeptides in STPs in northerntaly, showed in fact that some of them are not efficiently removednd end up in the receiving water. On average, 115–237 g of thesentibiotics per 1000 inhabitants are discharged every year by theTPs of the cities of Milan, Como and Varese. This correspondso a rough estimate of 7–14 tons of active principles dischargedvery year into the aqueous environment in Italy. Analysis of theeceiving rivers in northern Italy confirms these figures, with anverage load of 5 kg/day, or about 1.8 tons/year, of active principleound in the River Po at sampling sites covering a basin com-rising about one-fifth of the Italian population (i.e. an estimatedtons/year for the whole Italian population). The bulk of the antibi-tics contaminating the aqueous environment were quinolonesnd macrolides. Since prescriptions of these substances in Italy in008 were for a total of 70 tons [29], it means that up-to 10–20%f them was discharged in the environment after consumption.hile macrolides and quinolones contributed significantly to the

nvironment burden of antibiotics, other classes of antibiotics,uch as penicillins, were not found in significant amounts, despiteeing much more prescribed (320 tons/year in Italy in 2008) [29].herefore this paper give an important general indication, showinghat only some classes of antibiotics can be considered of con-ern for the environment and that a risk assessment might beocalized on particular classes of antibiotics but excluding otherlasses.

In conclusion, antibiotics, particularly macrolides anduinolones, are widespread environmental contaminants, andrban STPs are “hot spots” of their inflow in the environment.esults presented in this paper are from a case study carried outt a national scale level, with conclusions that can be generalizednd extended to other geographic areas.

cknowledgements

We thanks Mrs Judy Baggott for language help and writing assis-ance.

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Materials 179 (2010) 1042–1048

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