RESEARCH ARTICLE

Assessing the effects of tertiary treated wastewater reuseon the presence emerging contaminants in a Mediterraneanriver (Llobregat, NE Spain)

Rebeca López-Serna & Cristina Postigo & Juan Blanco & Sandra Pérez &

Antoni Ginebreda & Miren López de Alda & Mira Petrović & Antoni Munné &

Damià Barceló

Received: 15 March 2011 /Accepted: 25 August 2011# Springer-Verlag 2011

AbstractPurpose The Llobregat River, which is characterized byimportant fluctuations of the flow rates, receives treatedwaters from WWTPs. During the years 2007 and 2008, theLlobregat River basin suffered from a severe drought whichaffected the supply of drinking water facilities (DWF) thatrely on the exploitation of the river water. The Catalan

Water Agency implemented a water reuse experiment withthe objective of maintaining the river flow rate atsufficiently high level so as to ensure the supply of rawwater to Barcelona’s major DWF.Method A total of 103 emerging contaminants belonging tothe groups of pharmaceuticals (74), illicit drugs (17) andoestrogens (12) were determined using LC-MS/MS meth-ods in river water samples during the water reuse campaign.The effect of the reclaimed water discharge on the riverwater quality, in terms of contamination loads and environ-mental risk (based on the concentration addition mode), isdiscussed.Results Fifty-eight pharmaceuticals out of 74 monitoredwere detected at least in one sample. In river waterupstream (site R0) majority of compounds were detectedat low nanograms per litre levels, while downstream ofdischarge of tertiary effluent only few compounds weredetected at levels higher than 100 ng L−1 (i.e. acetamino-phen, diclofenac, erythromycin, sulfamethazine), but neverexceeding 500 ng L−1. The total concentration of illicitdrugs was found to be very low at both sampling sites(<50 ng L−1). No relevant ecotoxicity risks were identified,except for pharmaceuticals vs. algae.Conclusions In general, the discharge of reclaimed water inthe river influenced perceptibly in terms of concentrations,mass loads and environmental risk, especially for pharma-ceuticals and in less extent for illicit drugs. Nevertheless, itwas not very significant in any case.

Keywords Emerging contaminants . Pharmaceutical . Illicitdrugs . Oestrogens . LC-MS .Wastewater treatment .

Reclaimed water . Llobregat River .Mass balances . Riskassessment . Hazard quotients . Oestrogenic equivalents

Responsible editor: Henner Hollert

Electronic supplementary material The online version of this article(doi:10.1007/s11356-011-0596-z) contains supplementary material,which is available to authorized users.

R. López-Serna : C. Postigo : J. Blanco : S. Pérez (*) :A. Ginebreda :M. L. de Alda :M. Petrović :D. BarcelóDepartment of Environmental Chemistry, IDAEA-CSIC,c/Jordi Girona 18-26,08034 Barcelona, Spaine-mail: spsqam@idaea.csic.es

D. BarcelóCatalan Institute of Water Research,C/Emili Grahit, 101, Edifici H2O,Parc Científic i Tecnològic de la Universitat de Girona,17003 Girona, Spain

M. PetrovićInstitució Catalana de Recerca i Estudis Avançats (ICREA),Passeig Lluis Companys 23,80010 Barcelona, Spain

A. MunnéCatalan Water Agency,Provença 204-208,08036 Barcelona, Spain

D. BarcelóKing Saud University,P.O. Box 2455, 11451 Riyadh, Saudi Arabia

Environ Sci Pollut Res (2012) 19:1000–1012DOI 10.1007/s11356-011-0596-z

1 Introduction

Studies on water reuse are gaining relevance due to thegrowing water demand in agriculture and household.(Markus and Deshmukh 2010; Azar et al. 2010; Muñoz etal. 2009a; Aggeli et al. 2009; Xu et al. 2009; Wintgens et al.2008; Ghermandi et al. 2007; Dombeck and Borg 2005;Exall 2004). This is particularly important in areas whichsuffer from drought episodes and therefore their basinspresent important fluctuations in the flow rates likeLlobregat River (NE Spain). At the same time, thecatchment area is subject to heavy pressures ofcontamination coming from extensive urban, industrialand agricultural activities. During the period of 2007–2008, the Llobregat River basin suffered from a severedrought that caused severe problems of water scarcity.This situation seriously diminished the flow of the riverand therefore indirectly threatened the supply of drink-ing water in the region, especially in highly populatedareas like the city of Barcelona (Cazurra 2008; Mujeriego etal. 2008).

During a drinking water sources crisis, due to a severedrought that occurred in 2008, the water authority manag-ing water resources in the area of Barcelona consideredaugmenting drinking water supplies with reclaimed water.To know the possible environmental, health and waterquality effects of such water reuse, an experiment consist-ing of introducing reclaimed water in the river upstream ofthe potabilisation plant was performed during the autumn of2008 and 2009. The climatic and geographic scenarios ofsuch experiment, as well as the operational conditions, aredescribed in detail in the Supplementary information 1.This work deals with the second campaign (November2009), while the results of the previous one (autumn 2008)were already reported elsewhere (Köck-Schulmeyer et al.2010). All the experiments were monitored closely by theresponsible authorities in fulfilment of the existing regu-lations (Spanish Royal Decree 1620/2007). This refersbasically to conventional physical–chemical and microbio-logical contaminants, whereas the so-called emergingcontaminants are not yet covered therein. The aim of thepresent work was to monitor three classes of emergingcontaminants, namely pharmaceuticals (74), illicit drugs(17) and oestrogens (12), since many of these bioactivecompounds show an incomplete removal by conventionalwastewater treatment technologies (Barceló and Petrovic2007; Batt et al. 2007; Matamoros et al. 2009; Kasprzyk-Hordern et al. 2009; Pérez and Barceló 2008). Occurrencedata of these compounds at strategic sampling locations andat suitable time sites are described and compared with thework by (Köck-Schulmeyer et al. 2010). The resultspresented there, in that first campaign, pointed out that theeffect of reclaimed water discharge was not negligible in

terms of micro contaminants’ presence. But still, levelswere comparable or even below to those reported previ-ously in the Llobregat River and in other Spanish riversdischarging into the Mediterranean Sea.

As far as we know, this study, along with the onepresented by (Köck-Schulmeyer et al. 2010), are the firstpapers evaluating the effects of reclamation water toguarantee the availability of inlet water in a drinkingwater treatment plant, in terms of emerging contaminantsoccurrence.

Moreover, in order to get a proper interpretation of thefindings and to assess the effect of the tertiary effluent onthe receiving surface water quality from a quantitative pointof view, variability of both flows and concentrations wastaken into account by using load (mass flow) balances.Finally, a preliminary risk assessment for the concen-trations found in the river sampling sites was carried outfor the three groups of contaminants considered, throughthe estimation of their associated hazard quotients andoestrogenicity.

2 Materials and methods

2.1 Campaign description

2.1.1 Study site

The study site and the conditions under which theexperiment of releasing reclaimed water into the LlobregatRiver were run, are described in detail in Supplementaryinformation 1. The study was carried out in the last part ofLlobregat River (NE Spain) between the towns of Molinsde Rei (site R0) and Sant Joan Despí (site R2; Fig. 1). Thelatter is located ca. 8.5 km downstream at the inlet of animportant drinking water treatment plant, supplying waterto the city of Barcelona after appropriate potabilisationtreatment. The effluent of the tertiary treatment of theWWTP El Prat de Llobregat (site RW2) was pumpedupstream ca. 16.6 km and discharged into the river at ca.0.2 km below site R0.

2.1.2 Flow measurements

Flow measurements of the Llobregat River are regularlyperformed by the Catalan Water Agency (ACA) in thegauge flow station located in Sant Joan Despí (site R2).Measurements are recorded every 5 min and the dataaggregated at day intervals. They are accessible at the ACAwebsite (Agència Catalana de l'Aigua-Generalitat de Cata-lunya; http://www.gencat.cat/aca/). Tertiary effluent flowswere also available from the WWTP records. River flow atsite R0 (river upstream) QR0, though not measured, was

Environ Sci Pollut Res (2012) 19:1000–1012 1001

assumed to be the difference between that of site R2 (riverdownstream) QR2 and site RW2 (WWTP effluent), QRW2

since the distance between both sites was not too long, andno other significant water discharges and/or water abstrac-tions were located in that section of river. Therefore, underthe foregoing assumption the flow in site R0 could bereasonably estimated by subtraction to the flow value at siteR2 that of RW2.

Flow values for sites R0, R2 and RW2 corresponding tothe sampling dates, either measured (R2 and RW2) orcalculated (R0) are listed in Table 1.

2.2 Chemical analysis

2.2.1 Chemical reagents

Organic solvents were HPLC grade. Acetonitrile andmethanol were from Riedel de Häen (Steinheim, Germany).Target compounds (Tables S1, S2 and S3—Supplementaryinformation) were of high-purity grade (pharmaceutical>90%; drugs of abuse and oestrogens >96). In Supplemen-tary information 2, further information about the suppliersof the standards is presented.

Llobregat basinLlobregat basin

Fig. 1 Sampling pointslocation in the lower stretch ofthe Llobregat River. Point R0Llobregat River at Molins deRei, Point R2 Llobregat Riverat Sant Joan Despí; Point RW2discharge of tertiary effluentfrom WWTP El Prat deLlobregat

1002 Environ Sci Pollut Res (2012) 19:1000–1012

2.2.2 Sampling and sample preparation

The sampling campaign was carried out in November 2009.Water samples (5 L) corresponding to sites R0 and RW2(see Fig. 1) were collected in amber glass bottles, on sixdifferent days. Taking into account the flow rates and theexperience obtained during the 2008 campaign, grabsamples were taken at site R2 on the following day. Thetime elapsed between the two sampling events was deemedsufficient to allow the water sampled at R0 and RW2 toreach site R2. During the transportation of the samples tothe lab, they were kept in dark, cooled inside chambers withice and stored at −20°C until the analysis. Upon receptionin the lab, samples were immediately filtered through 1-μmglass fibre filters, followed by 0.45-μm nylon membranefilters (Whatman International Ltd, Maidstone, England),and stored in the dark at −20°C (in freezers) until analysis.

2.2.3 Analysis method

Sample preparation and preconcentration was performedaccording to previously reported validated methods by(López-Serna et al. 2010) for the analysis of the 74pharmaceuticals; by (Postigo et al. 2008) for the 17 illicitdrugs and by (Rodriguez-Mozaz et al. 2005) for theanalysis of the 12 oestrogens. All the methods consist ofonline SPE-LC-MS/MS. To control the performance of theanalytical methods for all the compounds, samples werespiked with specific isotopically labelled surrogate stand-ards prior to extraction as published previously. Themethodology for the determination of oestrogens wasmodified in that the number of isotopically labelledsurrogates was increased to six. Limits of detection andquantification for all target compounds are in Tables S4–S6(Supplementary information).

2.3 Load contributions and mass balances

For every compound i, and site of sampling j the load(expressed as mass flow in appropriate mass/time units)was computed as the product of concentration cij per flowQj: Hence, assuming a conservative behaviour for the

contaminants (see Section 3.2), for every compound i thefollowing mass flow balance equation holds:

QR2 � cR2i ¼ QR0 � cR0i þQRW2 � cRW2i ð1Þ

3 Results and discussion

3.1 Occurrence of micro contaminants

The occurrence of 103 emerging contaminants was studiedin both the Llobregat River (Fig. 1, sites R0 and R2) andthe effluents pumped upstream and discharged into the riverfrom the WWTP El Prat de Llobregat (Fig. 1, site RW2)after an adequate tertiary treatment (as described inSupplementary information 3). The results are summarizedin (Tables S1, S2 and S3 supplementary information).Generally, pharmaceuticals were found to be present athigher concentrations than illicit drugs and oestrogens inboth river water (Fig. 2a–c) and treated wastewater(Fig. 3a–c).

As far as pharmaceuticals are concerned (see Fig. 2a andTable S1 Supplementary information), 58 compounds out of74 monitored were detected at least in one sample. In riverwater upstream of discharge of tertiary effluent (site R0) 39compounds were detected at low nanograms per litre levels,while three analgesics and anti-inflammatory drugs (acet-aminophen, ibuprofen and salicylic acid) were the onlyones found at levels higher than 100 ng L−1, but rarelyexceeding 300 ng L−1. Other frequently detected com-pounds comprise anti-inflammatories diclofenac and nap-roxen, β-blockers atenolol and metoprolol, antibioticsofloxacin, erythromycin, clarithromycin and sulfamethox-azole and psychiatric drugs carbamazepine and lorazepam.However, in all cases concentrations detected were lowerthan 100 ng L−1.

In treated wastewater (site RW2) levels were higher,but still at <1,000 ng L−1 level for the majority ofcompounds (Fig. 3a). The exception were three antibiotics(sulfamethazine, azithromycin and erythromycin) and thediuretic furosemide that were detected at low microgramsper litre (max concentration found was 3.08 μg L−1 of

Site reference River upstream (R0) Riverdownstream (R2)

WWTPeffluent (RW2)

Dilution ratioQ RW2/QR2

Sampling datea QR0=QR2 − QRW2 QR2 QRW2

2–3/11/2009 3.71 4.77 1.06 0.22

5–6/11/2009 1.33 2.01 0.68 0.34

9–10/11/2009 1.7 2.47 0.77 0.31

12–13/11/2009 1.77 2.44 0.67 0.27

16–17/11/2009 1.74 2.62 0.88 0.34

19–20/11/2009 0.38 1.52 1.14 0.75

Table 1 Flow data (in cubicmetres per second) for thedifferent sites (R0: RiverLlobregat at Molins de Rei; R2:Llobregat River at Sant JoanDespí; RW2: WWTP tertiaryeffluent discharged)

a Sampling was carried out tak-ing into consideration the lagtime of the river water circula-tion between R0 and R2

Environ Sci Pollut Res (2012) 19:1000–1012 1003

0

500

1000

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R0 R2 R0 R2 R0 R2 R0 R2 R0 R2 R0 R2

2-3/11/2009 5-6/11/2009 9-10/11/2009 12-13/11/200916-17/11/200919-20/11/2009

Sampling point & date

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nce

ntr

atio

n (

ng

L-1

)

Analgesics and anti-inflammatories

Antibiotics

Antihypertensives

Antidiabetics

β agonists

β blockers

Barbiturates

Diuretics

Histamine H2 receptor antagonists

Lipid regulators

Psychiatric drugs

To treat cancer

To treat infections

0

10

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R0 R2 R0 R2 R0 R2 R0 R2 R0 R2 R0 R2

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Sampling point & date

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Amphenamine like compounds

Cannabinoids

Cocainics

LSD and metabolites

Opiates

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R0 R2 R0 R2 R0 R2 R0 R2 R0 R2 R0 R2

2-3/11/2009 5-6/11/2009 9-10/11/2009 12-13/11/2009 16-17/11/2009 19-20/11/2009

Sampling Point & date

Co

nce

ntr

atio

n (

ng

L-1

)

Conjugated estrogens

Free estrogens

a

b

c

Fig. 2 Concentrations (innanograms per litre) per familiesof compounds in river upstream(point R0) and downstream(point R2) to discharge thepoint. a Pharmaceuticals;b illicit drugs and c oestrogens

1004 Environ Sci Pollut Res (2012) 19:1000–1012

0

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L-1

)

Analgesics and anti-inflammatories

Antibiotics

Antihypertensives

Antidiabetics

β agonists

β blockers

Barbiturates

Diuretics

Histamine H2 receptor antagonists

Lipid regulators

Psychiatric drugs

To treat cancer

To treat infections

0

20

40

60

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140

2-3/11/2009 5-6/11/2009 9-10/11/2009 12-13/11/2009 16-17/11/2009 19-20/11/2009

Sampling date

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n (

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Amphenamine like compounds

Cannabinoids

Cocainics

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Opiates

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2-3/11/2009 5-6/11/2009 9-10/11/2009 12-13/11/2009 16-17/11/2009 19-20/11/2009Sampling date

Co

nce

ntr

atio

n (

ng

L-1

)

Free estrogens

Conjugated estrogens

a

b

c

Fig. 3 Concentrations (innanograms per litre) per familiesof compounds in tertiary WWTPeffluent (point RW2). a Phar-maceuticals; b illicit drugs andc oestrogens

Environ Sci Pollut Res (2012) 19:1000–1012 1005

furosemide in one sample) and several other compoundswere detected occasionally at levels between 100 and900 ng L−1 (codeine, salicylic acid, atenolol, enroflox-acine, ofloxacin, bezafibrate, fenofibrate and metronida-zole).

Observing concentrations in river water (site R0 up-stream of discharge and site R2 downstream of discharge oftertiary effluent; Fig. 2a), it is visible that the discharge ofthe tertiary effluent affects the levels of pharmaceuticals,but the increase in concentrations is not significant. At siteR2 only few compounds were detected at levels higher than100 ng L−1 (i.e. acetaminophen, diclofenac, erythromycinand sulfamethazine), but only the salicylic acid exceeds500 ng L−1.

In general, the compounds found at higher concen-trations and the proportion among them coincided withthe ones in (Köck-Schulmeyer et al. 2010), but levelswere higher in the present study. In fact, the totalconcentration of pharmaceuticals in the river upstream(RO) and downstream (R2) the discharge point, was two-and fourfold higher, respectively. In any case, levels werecomparable or even below to those reported previously forthe Llobregat River and in other Spanish rivers discharg-ing into the Mediterranean Sea (Farré et al. 2001; Kusteret al. 2008; Ginebreda et al. 2010; Muñoz et al. 2009b).

Concerning illicit drugs and their metabolites (seeFig. 2b and Table S2, Supplementary information), 10 outof the 17 investigated compounds were not detected in anyof the investigated samples. The whole families ofcannabinoids and LSD compounds were absent in waterphase, due to very low excretion rate and lypophilicity incase of cannabinoids and due to lower relative consump-tion (low individual dosage) in case of LSD. This fact wasalso observed in the study realized in 2008 (Köck-Schulmeyer et al. 2010). On the other hand, cocaine andbenzoylecgonine, and the amphetamine-like compoundephedrine were present in all water samples analysed,ephedrine being the one that was found at the highestconcentration, (79.5 ng L−1 in the tertiary effluent). It isinteresting to mention that cocaine and its major metab-olite benzoylecgonine presented higher average concen-trations in river upstream (site R0) than in dischargedtreated water (site RW2). The Llobregat River is subjectedto a heavy anthropogenic pressure, receiving extensiveurban and industrial waste water discharges. It receives theeffluent discharges of more than 55 WWTPs, and bothsampling sites (R0 and R2) are situated in the lower partof the river, which explains why levels of some contam-inants are higher in river water than in effluents of anefficient WWTP such is the case of WWTP El Prat.Regarding opiates, morphine was the only one detected,with a maximum concentration of 41.2 ng L−1 in treatedeffluent.

The impact of the tertiary discharge into the river waseven lower than the one observed for pharmaceuticals (seeFigs. 2b and 3b). Only small, not significant, increase inconcentrations is observed at sampling site R2 for somecompounds (ephedrine, ecstasy and metamphetamine),while for others the levels were lower. Generally, the totalconcentration of illicit drugs was found to be very low atboth sampling sites (<50 ng L−1). In fact, in contrast to thepharmaceuticals, the levels found in the present study, werelower than the ones presented by (Köck-Schulmeyer et al.2010), where, nevertheless, they did not surpass 100 ng L−1

in total. Also in terms of presence, the number of illicitdrugs detected was lower in the present study than inprevious campaign.

Regarding the oestrogens (see Fig. 2c and Table S3,Supplementary information), the positive findings corre-spond to oestrone, that was detected in both river water andtreated wastewater (see Figs. 2c and 3c) at levels neverexceeding 3 ng L−1, and diethylstilbestrol that was found atsampling site R0 (river upstream of discharge of tertiaryeffluent). Occurrence of diethylstilbestrol is especiallyinteresting since it is a banned substance, and its presenceis probably due to its fraudulent use as a growth promotantin farms. It was found only at sampling site R0 at averageconcentration of 4.5 ng L−1 and maximum level of12.2 ng L−1. These two compounds were also quantifiedin the study performed by (Köck-Schulmeyer et al. 2010) atconcentrations tenfold higher for the oestrone and atcomparable levels for the diethylstilbestrol. Also, in thepresent study, the number of oestrogens detected werelower, i.e. only two versus eight. The presence of those twooestrogens was also reported in the Llobregat tributary,Cardener River, by (Petrovic et al. 2002).

3.2 Load contributions and mass balances

To have a better insight on the relative influence of bothriver upstream and effluent discharge to the overallamount of pollutants found downstream in the river,direct comparison of concentrations is not adequate,since they do not take into account the respective flow.Instead, loads (expressed as mass flow in mass/timeunits) may provide a more convenient way to quantifyand compare the relative contribution of each stream(river and effluent). This is especially relevant, since oneof the objectives of the reuse experiment was to studythe effect of the dilution ratio of the reclaimed waterwith respect to the river in the overall pollution.Therefore, loads for sites R0, R2 and RW2 werecalculated as the product of concentrations per flowsfor the three groups of pollutants studied (Section 2.3).Compounds whose levels were below the limit ofdetection and/or quantification were set to zero.

1006 Environ Sci Pollut Res (2012) 19:1000–1012

It can be reasonably assumed that the load founddownstream (site R2) was only determined by the mixtureof the load coming from river upstream (site R0) with thatbrought by the effluent discharged (site RW2). Sincesampling site R0 and the discharge site RW2 were at veryclose distance (ca. 0.2 km) other influences in such a shortstretch may be considered negligible, thus allowingcomparison with their relative load contributions withoutmuch error. The results obtained at the different samplingdates and locations, expressed as percentages, are depictedin Fig. 4a–c for the three classes of compounds studied. Theeffluent contribution (RW2) seems to predominate over thatof the river upstream (R0) for both pharmaceuticals andillicit drugs, accounting for ca. 40–95% and ca. 50–90% ofthe total load, respectively. This contribution was slightlyhigher than the one reported in 2008 (Köck-Schulmeyer etal. 2010), probably due to lower effluent dilution ratios. Asexpected, for both classes of compounds, the highesteffluent contributions were observed with the lowesteffluent dilution (highest ratio QRW2:QR2=0.75), whichcorresponds to the last sampling date (see Table 1). Thatcan also explain that the contamination for the oestrogensthat day comes mainly from RW2 in contrast with the restof the sampling days, when the main contribution wascoming from the river. This is in sharp contrast with thepreceding campaign (2008) (Köck-Schulmeyer et al. 2010),in which oestrogens were exclusively traced to the effluent.Nevertheless, due to these analytes are scarcely detected,and when quantified, they are found at very low concen-trations (ca. 1–10 ng L−1), trustworthy conclusions cannotbe drawn.

Loads (mass flows) also provide a straightforward wayto compare in bulk the weight of the different pollutantfamilies studied in quantitative terms. The resultscorresponding to site R2 (river downstream), along thewhole sampling period, are summarized in Fig. 5. Fromthem, the following rank order was observed (concentrationranges are given in parentheses):

Pharmaceuticals (107–106 ng/s) >> illicit drugs (105–104 ng/s) > oestrogens (104–b.l.d. ng/s)

(b.l.d: below the limit of detection)

This order shows a clear predominance of pharma-ceuticals over the other classes, followed by illicit drugsand oestrogens, which is in full agreement with the rankfound in the study of 2008 (Köck-Schulmeyer et al.2010). To our knowledge, apart from that latter work andthe present one, no additional data regarding the loads ofthese emerging contaminants is currently available in theliterature.

Mass balances compliance was further checked upon theassumption of a conservative behaviour for the contami-

nants through the calculation of concentrations in site R2.This is a reasonable assumption given the short distancetravelled. Therefore, since both flow and concentrations atsite R2 (QR2 and cR2i) are already available from direct

Illicit Drugs Load Contribution

0%

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Estrogens Load Contribution

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Pharmaceuticals Load Contribution

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Fig. 4 Mass load relative contributions (%) of river upstream andtertiary effluent in point R2 at the different sampling dates. aPharmaceuticals; b illicit drugs and c oestrogens

Environ Sci Pollut Res (2012) 19:1000–1012 1007

measurements, the predicted value of cR2i(calc), which wasstraightforward calculated from the foregoing mass balanceequation (Section 2.3, Eq. 1) may be compared to theexperimental cR2i(exp). The suitability of these assumptionswas thus experimentally tested. Figure 6a and b show thegraphs obtained representing experimental vs. calculatedconcentrations according to the outlined procedure forpharmaceuticals and illicit drugs, for the different samplingdates. Even though there is an acceptable agreementbetween experimental and calculated values, the later tendto be overestimated, especially for the pharmaceuticals (thelinear regression coefficients together with the 99%confidence intervals are shown on the charts). In fact,76% of the concentration pairs are in this situation both forpharmaceuticals and illicit drugs. This could be due toseveral reasons, all of them attributable to deviations fromthe balance Eq. 1 related to the non-fulfilment of theimplicit assumption of conservative behaviour along theriver segment considered either of flows or concentrations.The following possibilities are suggested among others asplausible causes: (1) disappearance of the compound bysome kind of depletion mechanism occurring in the river,such as physical–chemical (e.g. photo degradation, adsorp-tion onto sediments, …) or biological degradation, and (2)by the possible existence of some flow alteration neglectedin the balance equation that might contribute to alter thedilution factor (QR2), such as depletion caused by infiltra-tion or, in the opposite sense, the occurrence of sometemporal minor water stream reaching the Llobregat Riverbetween sites R0 and R2. Whereas the second factor wouldbe expected to equally affect all compounds, the formerappears more likely since it was compound specific.However, no clear relations can be established betweenthe data of physico-chemical or biological degradationfound in the literature, and the overestimated resultsobserved. Thus, the epileptic drug, carbamazepine, is

known to be highly resistant to depletion, either by soiladsorption (Nakada et al. 2008; Scheytt et al. 2006; Chefetzet al. 2008), photo degradation (Chiron et al. 2006;Andreozzi et al. 2002) or biologically (Nakada et al.2006), but in the present study, it presents overestimationin 5 out of 6 days of sampling.

In any case, the differences between calculated andexperimental concentrations were in general very narrow,with relative standard deviations inferior to 40% in most ofcases, e.g. for carbamazepine, the average experimentalconcentration was 58 ng L−1 versus an estimated one of79 ng L−1.

In summary, the mass-flow balance equation mayacceptably serve to predict downstream concentrations ofpollutants if flows and concentrations corresponding to bothriver and effluent discharges occurring upstream are known,and the distance travelled by water is moderate.

3.3 Ecotoxicological and oestrogenic assessment

Adverse ecotoxicological effects caused by pollutants onaquatic organisms are the main problem of their presence inthe environment. The so-called hazard quotient (HQhereafter) is a suitable tool to estimate the ecological riskattributable to each pollutant (Kim et al. 2007). The WaterFramework Directive (Sanderson et al. 2003), stated theconvenience of assessing taxa belonging to three differenttrophic levels. Commonly, algae, daphnids and fish are usedas reference organisms. Whereas EMEA guideline proposesthe calculation of this quotient as the ratio betweenPredicted Environmental Concentrations (PEC) and Pre-dicted No-Effect Concentration (PNEC) (Grung et al.2008); (Huschek et al. 2004), other authors prefer usingMeasured Environmental Concentrations (MEC) instead ofPEC (Santos et al. 2010). Hazard quotients defined as HQ =MEC/PNEC are determined for every compound present on

1.00E+00

1.00E+01

1.00E+02

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

R0 R2 R0 R2 R0 R2 R0 R2 R0 R2 R0 R2

2-3/11/2009 5-6/11/2009 9-10/11/2009 12-13/11/2009 16-17/11/2009 19-20/11/2009

Sampling point & date

Lo

ad (

ng

s-1

)

Pharmaceuticals

Illicit drugs

Estrogens

Fig. 5 Comparison of massloads (nanograms per second)per compound classes in pointsR0 (river upstream) and R2(river downstream) at thedifferent sampling dates

1008 Environ Sci Pollut Res (2012) 19:1000–1012

the scenario under study. However, since PNEC data are notalways available in the literature, acute toxicity values arecommonly utilized. Thus, PNECs are typically calculatedfrom EC50 values corrected by a safety factor of 1,000 as itis recommended by the Water Framework Directive(Directive 2000/60/EC). The so calculated HQ are thusequivalent to the often used toxic units (TU) (Sprague andRamsay 1965), differing only on the correcting safetyfactor. Irrespectively whether HQ or TU are used, they canbe aggregated by simple addition on an overall hazardquotient (HQ = ∑HQi) under the assumption of theconcentration addition (CA) mode of action (Bliss 1939).CA is based on the assumption of a similar mode of actionfor all the substances present in the mixture. This impliesthat all components contribute to the final effect even at lowconcentrations or said in other words that concentrationscan be added. Based on solid experimental support there isa wide consensus on accepting CA as a general purpose

model of choice for a first approximation to mixturetoxicity estimation (Backhaus 2011). Nevertheless, it mustbe reminded that synergic effects among compounds cantake place (Santos et al. 2010), so the sum of single hazardquotients must be interpreted as a first rough approach.Upon that basis, it is usually accepted that if HQ is equal orhigher than 1 potential impairment of the aquatic ecosystemis likely to occur. Ecological risks caused by micropollutants are expected to be related with chronic toxicity.Hence, in order to take into account that chronic toxicitymight occur at lower concentrations than the acute one, thatsafety factor of 1,000 was applied in the calculation ofthose PNEC. In the present work, hazard quotients havebeen calculated individually for the minimum and themaximum concentration found of every pharmaceutical andillicit drug compound, in river sites R0 and R2. Afterwards,a total HQ was estimated, by adding all those individualvalues for both groups of micro contaminants. Experimen-

PHARMACEUTICALSCalculated vs. Experimental concentrations at R2

y = 0.74x + 0.5113

R2 = 0.6916

-2

-1

0

1

2

3

4

-2 -1 0 1 2 3 4

Log experimental concentrationL

og

cal

cula

ted

co

nce

ntr

atio

n

Regressionline

Superior limit

Inferior limit

Ideal line

ILLICIT DRUGSCalculated vs. Experimental concentration at R2

y = 0.7699x + 0.20691

R2 = 0.5903

-0.75

-0.25

0.25

0.75

1.25

1.75

2.25

2.75

3.25

-0.75 -0.25 0.25 0.75 1.25 1.75 2.25

Log experimental concentration

Lo

g c

alcu

late

d c

on

cen

trat

ion

Regressionline

Superior limit

Inferior limit

Ideal line

a

b

Fig. 6 Calculated vs. experi-mental concentrations in pointR2 for a pharmaceuticals andb illicit drugs

Environ Sci Pollut Res (2012) 19:1000–1012 1009

tal EC50 values extracted from the literature were preferred,but when they were not available, EC50 were estimated usingUSEPA’s ECOSAR (Ecological Structure Activity Relation-ships) model (ECOSAR v1.00). When more than one EC50

value was available, the lowest was taken into consideration(Grung et al. 2008). Therefore, the risk in the most extremesituation has been in any case considered. In Tables S7 andS8 (Supplementary information), the values of EC50 for fish,daphnid and green algae used for the calculation of PNEC arereported.

In what pharmaceuticals are concerned, only two out ofthe 74 compounds studied (ciprofloxacin and sulfamethox-azole) showed an individual HQ above 1, and in all casesthat toxicity corresponded to algae. It was attributable to thelow EC50 of the foresaid two compounds (ciprofloxacin0.005 mg L−1 and sulfamethoxazole 0.027 mg L−1; seeTable S7, Supplementary information), which give rise tothe corresponding HQ values as high as 15.6 in site R2 and2.9 in R0 for ciprofloxacin, or 4.6 and 1.9 in R2 and R0 forsulfamethoxazole. For the other two reference species(daphnids and fish), individual HQs were below 1 in allsites of sampling. As regards as illicit drugs, all theindividual HQs for the different compounds were far under1. Thus, even upon aggregation of all the HQs for everycompound, global HQ values obtained would be clearlyunder that limit for all the species assessed.

Hence, and as it can be observed in the charts shown inFig. 7a, b, the environmental risks based on HQs in R0 andR2 are in general low for pharmaceuticals and negligiblefor illicit drugs. Pharmaceuticals vs. algae was the onlyexception for which the total HQ, considering the contri-bution of all the pharmaceuticals assessed altogether,resulted with maximum values of ca. 22 and 5 in R2 andR0, respectively. The overall relative order of susceptibilityto pharmaceuticals was estimated to be green algae >>daphnid > fish in river both sites R0 and R2, whereas forillicit drugs (see Fig. 7b), the order observed was daphnid ≈green algae > fish, being the differences among organismsless pronounced than for pharmaceuticals. HQ ranges forboth pharmaceuticals and illicit drugs are slightly higher insite R2 than in site R0, though of the same order ofmagnitude.

Overall oestrogenicity was also determined in the sameriver sites R0 and R2 by addition of the individualoestrogenic activities of each compound. They werecalculated as estradiol equivalent concentration (EEQ)multiplying the oestrogenic potency for every compoundby its concentration. The oestrogenic potency was thusgiven as estradiol equivalency factors (EEFs), defined asEC50 of the compound relative to the EC50 of 17β-estradiol. In Table S9 (Supplementary information), theEEFs for the free oestrogens are shown. Values forconjugated ones were not found in the literature. When

more than one value was found for the same oestrogen, thehighest one was chosen in order to assess the worstsituation possible. The oestrogenic activity of the analysedriver waters (R0 and R2) is shown in Fig. 8 for theconcentration range of two oestrogens quantified (oestroneand diethylstilbestrol). Although the levels of concentrationfound were quite low, they may represent some risk sinceoestrogenic effects in aquatic ecosystems have beenreported at levels of estradiol as low as 1 ng L−1 (Hansenet al. 1998; Purdom et al. 1994). In the case presented,EEQs of up to 1.10 and 13.42 ng L−1 were determined insite R0 (river upstream) for oestrone and diethylstilbestrol,respectively. Individual values calculated for site R2 wereunder 1 ng L−1 in all cases.

4 Conclusions

In the present work, the impact of wastewater reuse on theriver water quality has been examined by monitoring theoccurrence of three classes of emerging contaminants,namely pharmaceuticals, illicit drugs and oestrogens. Thisstudy entails the second campaign, thus completingpreliminary findings of the preceding one carried out in2008 and allowing to compare results and to consolidateconclusions gathered from both campaigns.

0

5

10

15

20

25

R0 R2 R0 R2 R0 R2

Fish Daphnids AlgaePharmaceuticals

Sampling Point & Organism

Haz

ard

Qu

oti

ent

0

0.005

0.01

0.015

0.02

R0 R2 R0 R2 R0 R2

Fish Daphnids AlgaeIllicit Drugs

Sampling Point & Organism

Haz

ard

Qu

oti

ent

a

b

Fig. 7 Hazard quotients for pharmaceuticals at points R0 (riverupstream) and R2(river downstream) for the different organisms: apharmaceuticals and b illicit drugs

1010 Environ Sci Pollut Res (2012) 19:1000–1012

As far as pharmaceuticals are concerned, comparingconcentrations in river water upstream and downstream tothe site of discharge of the reclaimed tertiary effluent, itseffect, the increase in concentrations, though perceptible isnot significant. Regarding illicit drugs and their metabo-lites, the impact of the discharge into the river was evenlower than the one observed for pharmaceuticals. In turn, incontrast with the previous campaign, oestrogen levels wereclearly higher upstream than downstream of the river.

A complementary approach aiming to distinguish theorigin of the overall pollution found in the river down-stream was attempted through the comparison of loads(mass flows). Whereas pharmaceuticals and illicit drugsloads were mostly allocated to the effluent, the origin ofoestrogens was mainly river upstream.

Moreover, assuming mass conservation behaviour,the mass balance equation also allowed to roughlyestimate the concentration of the emerging compoundsdownstream.

Finally, an ecotoxicity risk assessment comparing riverupstream and downstream to the discharge site wasperformed for pharmaceuticals and illicit drugs for threereference organisms belonging to different trophic level(fish, dapnids and algae). Again, differences between riverupstream and downstream to the discharge site wereperceivable, but not very significant. Overall, no relevantrisks were identified, with the only exception of pharma-ceuticals vs. algae that were likely to occur both in riverupstream and downstream. Such algae toxicity was mostlyattributable to the extremely low EC50 reported for twocompounds, i.e. ciprofloxacin and sulfamethoxazole. Oes-trogenicity was similarly evaluated using estradiol equiva-lent factors (EEQs) for the oestrogenic compoundsidentified. In the present case river upstream showed higheroestrogenicity than river downstream, attributable to oes-trone and diethylstilbestrol.

Acknowledgements This work has been supported by the CatalanWater Agency, the Spanish Ministry of Science and Innovation[projects Cemagua CGL2007-64551/HID and Consolider-Ingenio2010 Scarce CSD2009-00065]. Merck is acknowledged for the giftof LC columns and Spark Holland for the gift of online SPEcartridges. RLS acknowledges the Spanish Ministry of Educationand Science for the economical support through the FPI pre-doctoralgrant. CP acknowledges AGAUR (Generalitat de Catalunya, Spain)for their economical support through the FI pre-doctoral grant. SPacknowledges the contract from the Ramón y Cajal Program of theSpanish Ministry of Science and Innovation.

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