Chemosphere 89 (2012) 164–171

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Chemosphere

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Mass balance of pharmaceutical and personal care products in a pilot-scalesingle-sludge system: Influence of T, SRT and recirculation ratio

Sonia Suárez ⇑, Rubén Reif, Juan M. Lema, Francisco OmilSchool of Engineering, University of Santiago de Compostela, Rue Lope Gomez de Marzoa s/n, 15705 Santiago de Compostela, Galicia, Spain

h i g h l i g h t s

" We study the fate of 16 pharmaceutical and personal care products." Accurate mass balances are applied in a single-sludge pilot scale plant." Volatilization, biodegradation and sorption is quantified." Influence of temperature, sludge, recirculation and redox conditions is evaluated.

a r t i c l e i n f o

Article history:Received 2 March 2012Received in revised form 24 May 2012Accepted 25 May 2012Available online 23 June 2012

Keywords:BiotransformationPPCPsRemovalSorptionWastewater

0045-6535/$ - see front matter � 2012 Elsevier Ltd. Ahttp://dx.doi.org/10.1016/j.chemosphere.2012.05.094

⇑ Corresponding author. Tel.: +34 881 816 740; faxE-mail addresses: sonia.suarez@usc.es (S. Suárez)

juan.lema@usc.es (J.M. Lema), francisco.omil@usc.es (

a b s t r a c t

The influence of operation condition on the fate of 16 pharmaceutical and personal care products (PPCPs)in a single-sludge nitrifying/denitrifying pilot plant was assessed. Volatilisation, sorption and degradationwere included in the mass balances to determine the most relevant removal mechanisms during PPCPtreatment.

Sludge retention time (SRT) was an important factor for the removal of compounds that significantlysorb onto sludge, as ethinylestradiol, whose removal increased 11% when working at SRT above 20 d.The internal recirculation ratio was significant for the removal of moderately biodegradable compounds,as citalopram. The positive effect of operating at warmer temperatures was particularly significant fortwo antibiotics, implying a 30% increase in their transformations. In the case of naproxen, an influenceof sludge acclimation and concentration was observed, leading to removal efficiencies from 27% to 99%.

Concerning removal mechanisms, most compounds were removed due to biotransformation, althoughfor fragrances sorption and volatilisation played a role.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

During the last decade, the main focus when trying to improvethe quality of water has gradually shifted from conventional pollu-tants (organic matter, solids and nutrients) to more specific xeno-biotic compounds. As some of these substances are detected at theng L�1 up to the lg L�1 level, they are described as micropollutants.These include aromatic hydrocarbons, sulphonated compounds,heavy metals and, more recently, pharmaceuticals and personalcare products (PPCPs), whose occurrence in urban wastewatersfrom all over the world is demonstrated nowadays (Ternes, 1998;Stumpf et al., 1999; Carballa et al., 2004; Nakada et al., 2006). Con-tamination of the aquatic media, mainly surface and ground water,depends strongly on the removal efficiency achieved in sewagetreatment plants (STPs), which were not designed for the complete

ll rights reserved.

: +34 881 816 702., ruben.reif@usc.es (R. Reif),F. Omil).

elimination of these type of substances (Bester, 2004; Kupper et al.,2006; Gomez et al., 2007). In fact, the direct relation that exists be-tween the presence of PPCPs in surface water and the discharge ofSTP effluents has been evidenced in several works (Stumpf et al.,1999; Heberer et al., 2002). This is of special concern when the pro-portion of the discharge is significant with respect to the naturalwater flow. Moreover, organisms living in effluent-dominated sys-tems are subjected to chronic exposure to complex mixtures ofPPCPs which could potentially cause undesirable effects (Brookset al., 2006).

Regarding STP design and operation, it is thought that parame-ters such as hydraulic retention time (HRT), sludge retention time(SRT), redox conditions and temperature affect the removal ofPPCPs. Concretely, the SRT determines the mean residence timeof microorganisms inside the reactor. Consequently, only organ-isms which are able to reproduce themselves during this timecan be retained and enriched in the system. According to this def-inition, high SRTs allow the enrichment of slowly growing bacteriaand consequently, the establishment of a more diverse biocoenosis

S. Suárez et al. / Chemosphere 89 (2012) 164–171 165

with broader physiological capabilities. For several PPCPs a posi-tive effect on their removal has been observed when working athigher SRTs and a critical value for this parameter of 10 d was iden-tified (Clara et al., 2005a). Regarding redox conditions and temper-ature, differences in the removal efficiencies for some PPCPs havealso been reported (Joss et al., 2004). For example, Vader et al.(2000) found that during a biological treatment process the re-moval of ethinylestradiol (EE2) stopped when the sludge lost itsnitrification capacity due to low temperatures. In general, the roleof temperature is relevant for microbial activity, since it stronglyinfluenced microorganism growth rate (Price and Sowers, 2004).

The vast majority of data published in the field of PPCP removalfrom wastewaters refer to full-scale STPs, in which the raw influentand final effluent were sampled in order to determine the overallremoval of PPCPs from the liquid phase. In some works, the impor-tance of sorption and/or volatilisation in PPCP removal was consid-ered (Bester, 2004; Joss et al., 2004, 2005; Clara et al., 2005a;Kupper et al., 2006). Other authors analysed the differences in PPCPelimination during primary and secondary treatment (Carballaet al., 2004; Kupper et al., 2006). Additional information aboutthe behaviour of PPCPs in biological lab- and pilot-scale plants isalso available, although much less frequent (Clara et al., 2004a,2005a; Suarez et al., 2010).

The aim of the present work was to perform a long-term studyon the fate and behaviour of 16 PPCPs in a predenitrifying activatedsludge pilot plant. During the 3 yr of bioreactor operation, anextensive sampling campaign including the different streams ofthe system was carried out so as to evaluate the influence of redoxconditions (anoxic and aerobic), temperature, recirculation flowfrom the aerobic to the anoxic tank, sludge concentration andSRT on the transformation of selected PPCPs.

2. Materials and methods

2.1. Single-sludge treatment plant

The experimental equipment used is a single-sludge system di-vided into a first anoxic and a second aerobic zone, supplied with asecondary sedimentation tank (Fig. 1). The total useful volume ofthe reactor is 30 L, of which 40% correspond to the anoxic fractionand the rest to the aerobic compartment. The fraction of anoxicvolume was selected on the basis of average design criteria offull-scale pre-denitrifying systems and taking into account the spe-cific COD and N balance of this pilot plant. It is habitual to mini-mise aerobic tank volumes in order to reduce operational costs if

P-2

Water

O

Anoxic Tank

P-3

P-1

Concentrate

Rext

Rint

Feed

Fig. 1. Schematic diagram of the

complete nitrification is guaranteed. An external recirculationstream returns the biomass retained in the settler to the inlet ofthe plant at a ratio of 0.5 compared to inlet flow. Nitrate recircula-tion from the aerobic to the anoxic tank was carried out at a ratioinitially set at 3, but increased to 4 after 10 months of operation inorder to enhance the removal efficiency of nitrogen. In this type ofsingle-sludge configuration, biomass is composed of an associationof autotrophic and heterotrophic microorganisms that are acti-vated alternately according to the environmental conditions ofthe two compartments.

The reactor was fed with a synthetic medium that reproducesthe chemical characteristics of a medium charged urban wastewa-ter (in mg L�1: 500 of COD, 40 of NH4–N and 8 of PO4–P) which wasspiked with PPCPs at concentrations between 10 and 40 lg L�1

(Supplementary material (SM), Table SM-1).During the start-up period, which lasted 3.5 months, the SRT

was maintained at 30 d and controlled by daily sludge purgesand no PPCPs were spiked to the feed. During this period, a diver-sified biota developed in the bioreactor, including slowly-growingbacteria, and stable operational conditions were achieved. TheHRT, composition of feed and dissolved oxygen level were main-tained constant during the whole process. Temperature was notcontrolled and varied according to the indoor temperature, whichallowed the differentiation of two operation periods: 14–18 �C dur-ing autumn and winter and 18–23 �C during spring and summer.The SRT varied between <20, 20–40 and >40 d, due to the fluctua-tions of volatile suspended solids (VSS) concentrations inside thepilot plant or due to sludge purges.

The selected PPCPs were regularly monitored along the pilotplant. The first group of pharmaceuticals added to the reactor com-prised (a) anti-inflammatory drugs (ibuprofen (IBP), naproxen(NPX) and diclofenac (DCF)), (b) neutral compounds (carbamaze-pine (CBZ) and diazepam (DZP)), (c) anti-depressants (fluoxetine(FLX) and citalopram (CTL)) and (d) hormones (EE2 and estradiol(E2)). Then, the number of PPCPs considered in this research wasbroadened by incorporating (e) antibiotics (erythromycin (ERY),roxithromycin (ROX), sulfamethoxazole (SMX) and trimethoprim(TMP)) and (f) fragrances (galaxolide (HHCB), tonalide (AHTN)and celestolide (ADBI)), which were monitored for 18 months.

2.2. Analytical methods

Soluble COD, Total and VSS, ammonium, nitrite and nitrate con-centrations were determined following Standard Methods (APHA,1999). Analyses of PPCPs were performed after collecting 1-L grab

Air

Sedimentation Tank

D pH

Aerobic Tank

P-4

Sludge Purge

T

EffluentSed in

activated sludge pilot plant.

166 S. Suárez et al. / Chemosphere 89 (2012) 164–171

samples in glass vessels from the anoxic and aerobic tanks. Addi-tionally, samples from the feed, effluent and external recirculationwere collected during 1 h in aluminium bottles. The samples wereimmediately prefiltered (AP4004705, Millipore, 0.7 lm) andsupplemented with a pinch of sodium azide (�0.3 g L�1). For theanalysis of PPCPs, sample extraction based on Solid Phase Extrac-tion or Solid Phase MicroExtraction was used as a pre-concentra-tion technique prior to their quantitative determination. Liquidor Gas Chromatography coupled to Mass Spectrometry (LC–MS orGC–MS, respectively) was used for the final quantification. Moredetailed information about the different methodologies used forPPCP quantification is provided in Suarez et al. (2010).

2.3. Mass balances calculations

The main removal processes to be considered for PPCPs duringtheir passage through the pilot plant are stripping, sorption andbiological degradation.

Due to the intensive aeration in the aerobic compartment, strip-ping could be a removal pathway for PPCPs with a high Henry’scoefficient (H), being the mass flow of compound j removed by vol-atilisation (Fj,Stripped in lg d�1) calculated as follows:

Fj;Stripped ¼ Cj;airqairQ ¼ HCj;dissAerqairQ ð1Þ

where Cj,air is the concentration of compound j in the gas phase(lg L�1

air ), Q the flow rate treated in the pilot plant (L d�1) and qair

the aeration applied per unit of wastewater treated (Lair L�1wastewater).

If an equilibrium described by Henry’s law is assumed, concentra-tion in the gas phase can be related with the concentration of com-pound j in the liquid phase of the aerobic compartment (Cj,diss Aer inlg L�1). Since H is below 10�6 for all the selected substances exceptfor fragrances, the influence of volatilisation was only evaluated forthese compounds.

The fraction of compound sorbed to sludge is estimated assum-ing sorption equilibrium (Ternes et al., 2004a) according to:

Kd;j ¼Cj;sorbed

Cj;dissSSTið2Þ

where Kd,j is the solid–water distribution coefficient of compound j(L kg�1), Cj,sorbed is the concentration of compound j sorbed ontosludge (lg L�1), SSTi the suspended solids concentration in streami (kg L�1) and Cj,diss the dissolved concentration of compound j(lg L�1).

In the case of musk fragrances, Kd,j was determined experimen-tally for the biological sludge developed in the pilot plant of thiswork, by means of measuring the total and the soluble concentra-tions of musk compounds in the mixed liquor. The other Kd,j valuesapplied in the mass balances were taken from bibliography (TableSM-1). Priority was always given to experimentally determinedvalues, although in the case of the two anti-depressants (FLX andCTL) this parameter had to be estimated from their KOW, followingthe procedure described in Jones et al. (2002).

The total mass flow of compound j in stream i (Fj,i), is the sum ofthe amount present in the liquid phase (Fj,iLiq) and the fractionsorbed on sludge particles (Fj,iSol):

Fj;i ¼ Fj;iLiq þ Fj;iSol ¼ Cj;iQ i þ Q iKd;jSSTiCj;i ¼ Cj;iQ ið1þ Kd;jSSTiÞ ð3Þ

where Qi is the flow rate of stream i (L d�1).Total mass flows for each trace pollutant were determined for

the feed, internal recirculation (Rint), external recirculation (Rext),sludge purge, inflow of the settler (Sedin) and effluent (Fig. 1).The difference between the total flow of compound j that entersand leaves one compartment of the pilot plant, including volatilisa-tion in the aerobic tank, can be attributed to biological transforma-tion if steady state conditions are assumed (Eq. (4)):

Ej;Anox ¼ðFj;FeedþFj;RintþFj;RextÞ�Fj;Anox out

Fj;Feed100

Ej;Aer ¼Fj;Anox out�ðFj;Sed inþFj;Rint Þ�Fj;stripped

Fj;Feed100

Ej;Sed ¼Fj;Sed in�ðFj;EffluentþFj;RextþFj;PurgeÞ

Fj;Feed100

) Ej;Plant ¼Fj;Feed�ðFj;EffluentþFj;PurgeÞ�Fj;Stripped

Fj;Feed100

ð4Þ

where Ej,Anox, Ej,Aer and Ej,Sed are the removal efficiencies for com-pound j in the anoxic, aerobic and sedimentation tank, respectively,and Ej,Plant the global efficiency of the plant (%). The term (Fj,Feed +Fj,Rint + Fj,Rext) constitutes the influent to the anoxic tank of the pilotplant (Fj,Anox in), whereas Fj,Anox out represents the outflow from thistank. Similarly, (Fj,Sed in + Fj,Rint) is the outlet of the aerobic tank(Fj,Aer out) and (Fj,Effluent + Fj,Rext + Fj,Purge) the total discharge of the sed-imentation tank (Fj,Sed out). In the mass flow of the feed (Fj,Feed inlg L�1) sorption was considered negligible, since the synthetic feeddid not contain any solid particles.

Biological transformation of PPCPs can be described by pseudofirst-order kinetics (Joss et al., 2006):

rS ¼ �dCj;total

dt¼ kbiolVSSCj;diss ð5Þ

where rS is the reaction rate (lg L�1 d�1), Cj,total the total concen-tration of compound j (lg L�1), t the time (d), kbiol is the reactionrate constant (L g�1 d�1) and VSS the volatile suspended solids con-centration (g L�1).

For estimating kbiol for the selected PPCPs the process was ap-proached to a steady-state, continuous stirred-tank reactor model:

Fj;Feed � ðFj;Eff þ Fj;Purge þ Fj;StrippedÞ � rSV ¼ 0 ð6Þ

where V is the reaction volume (L).

3. Results and discussion

3.1. Conventional operation parameters

Dissolved oxygen concentration in the aerobic compartmentwas maintained as high as possible during the first 10 months inorder not to limit the development of a stable nitrifying biomassin the aerobic reactor. Afterwards it was lowered to dissolved oxy-gen concentrations in the range of 2.5–4.5 mg L�1. Simulating thenormal situation in full-scale plants, temperature was not con-trolled, and the fluctuations observed are due to ambient temper-ature variations. However, this fluctuation is softened since theplant was situated indoors (14–18 in winter and 18–23 �C insummer).

Biomass concentration was followed along the sampling pointsof the pilot plant. The reactor was inoculated with 2 g VSS L�1 ofsludge, which duplicated due to bacterial growth after approxi-mately 140 d and remained quite constant at 4 g VSS L�1 untilday 590. After a period of around 130 d of decrease in the sludgeconcentration inside the reactor, it stabilized again at around2 g VSS L�1. This decrease in the concentration of sludge was asso-ciated to a low biomass growth rate due to the operation at highSRT. Additionally, the sludge presented poor settleability due tothe development of filamentous bacteria which led to high losseswithin the effluent.

The concentration of COD and nitrogen in the form of ammonia,nitrite and nitrate was also followed along the pilot plant. Overallremoval efficiencies stabilized 10 d after the start-up of the plant at�82% for nitrogen and �95% for COD. Once the internal recircula-tion ratio was raised from 3 to 4 (at day 323), nitrogen removalefficiency increased to values above 90%.

S. Suárez et al. / Chemosphere 89 (2012) 164–171 167

3.2. Fate of PPCPs in the bioreactor

Table SM-2 summarises the removal rates considering thewhole set of influent and effluent concentrations of PPCPs in theliquid phase. IBP and NPX removal was very effective (>80%), ingood agreement with disappearances previously observed in acti-vated sludge full-scale STPs of Germany, Spain, England or Japan(Ternes, 1998; Nakada et al., 2006; Gomez et al., 2007; Joneset al., 2007).

DCF, CBZ and DZP removals were below 20%. In the case of CBZthe low removal percentage achieved during treatment in the pilotplant was consistent with previously reported data (Ternes, 1998;Joss et al., 2005), which could make it suitable as marker foranthropogenic influences on the aquatic environment (Claraet al., 2004b). Removal data for DZP during biological treatmentare scarce, probably because this compound is normally not de-tected in raw wastewaters (Carballa et al., 2004; Clara et al.,2005b). However, some authors (Kreuzinger et al., 2004; Castigli-oni et al., 2006) have reported removal efficiencies ranging from�5% up to 25% during sewage treatment. Analysing the Kd values(Table SM-1) and biodegradation kinetics (kbiol < 0.1 L g SS�1 d�1

according to Joss et al. (2006)) reported for this compound, neithersorption nor degradation is expected to be significant, which wasactually confirmed in the studied pilot plant. Contradictory resultsabout DCF removal can be found in literature, for which reportedremovals vary from 0% to 75% (Clara et al., 2005b; Gomez et al.,2007). Considering that biological degradation is governed bypseudo-first order kinetics and that the degradation constantdetermined for this pharmaceutical is below 0.1 L g SS�1 d�1 (Josset al., 2006), the half life of this pharmaceutical for plants workingwith a biomass concentration in the range of 2–4 g L�1 can be esti-mated as 2–3.5 d. This means that only those plants operating athigh HRT are expected to be able to degrade it to a certain extent,since DCF is not expected to be significantly retained in the reactorby sorption (Kd 1.2 L kg�1). This dependency of DCF removal withthe HRT of the plant can be observed in Clara et al. (2005b), whoreported 70% removal of DCF for a STP working at a HRT of 13 d,whereas this efficiency was reduced to negligible removals forthe other two STPs which worked at HRT < 1.2 d. It also explainsthe low removals achieved in this research in which the pilot plantwas operated at a HRT of 1 d.

Elimination reached in the pilot plant for estrogens was 97–98%and 78–81% for E2 and EE2, respectively. Similar data have been re-ported previously, with removals between 85% and 100% for E2

Table 1Summary of the results obtained in the present work, including biological transformationinfluence of the different operational conditions.

Compound kbiol Transformation

Anoxic Aerobic

CBZ <0.07 �� ��DZP <0.04 �� ��DCF <0.1 �� ��FLX 1.6 ± 1.0 ��/+ �/++NPX 3.3 ± 2.8 �� ++IBP 3.7 ± 2.8 �� ++E1 + E2 32 ± 6 �� ++EE2 1.0 ± 0.1 �� +/++CTL 0.41 ± 0.01 �� �+SMX 0.60 �� �+/+ROX 1.7 �� +/++TMP 0.65 ��/�+ ��/+ERY 2.4 �/�+ �/+HHCB 4.1 ± 2.2 �/+ �/�+AHTN 4.0 ± 1.8 -+/++ –/-+ADBI 3.5 ± 3.2 �/�+ ��/�

(��) < 20; (�) 20–40; (�+) 40–60; (+) 60–80; (++) > 80%. The influence of sludge retransformation degree is indicated as yes, no or not analysed (n.a.).

and in the range of 70–95% for EE2 (Baronti et al., 2000; Josset al., 2004; Nakada et al., 2006).

From the two anti-depressants considered, FLX was found to bebetter removed than CTL, with efficiencies of 71–85% and 28–50%,respectively. For FLX the biodegradation profile was estimated tobe >91% by Webb (2004), whereas Johnson et al. (2005) estimatedremovals for both anti-depressants of �20%. Vasskog et al. (2006)found efficiencies in the range of 8–70% for FLX and 29–57% forCTL during one sampling campaign of the influent and effluent ofthree STPs in Norway.

Antibiotics (ROX, ERY, SMX and TMP) removal reached values of64–70% which are considerably higher values than data reportedfor the same antibiotics in literature. For ROX maximum removalpercentages of 40% have been reported by Göbel et al. (2007) andJoss et al. (2005). ERY and TMP showed high persistence duringwastewater treatment in several full-scale monitoring campaigns(Lindberg et al., 2005; Castiglioni et al., 2006; Göbel et al., 2007).In the case of SMX, eliminations in the range of �138% up to 71%have been found in previous works (Carballa et al., 2004; Josset al., 2005; Lindberg et al., 2005; Castiglioni et al., 2006; Göbelet al., 2007). It is worth noting that the cited references report rem-ovals of antibiotics in full-scale STPs that have been monitoredduring a specific time period, whereas a controlled synthetic med-ium was used in the present work. More homogenous data areexpected in this work since processes occurring at full scale, ascleavage of conjugates, presence of metabolites, release of macro-lides enclosed in faeces particles, etc. are avoided.

The fragrances considered in the present work (HHCB, AHTNand ADBI) were very effectively removed from the liquid phase(�90%), in concordance with the performance observed in differentSTPs across Europe and the USA (Simonich et al., 2002; Kupperet al., 2006), although somewhat higher than removals reportedin Bester (2004) or Carballa et al. (2004) during biological treat-ment. When primary treatment is included, the overall removalof HHCB and AHTN measured in Carballa et al. (2004) reach thelevels presented in this work. According to the high lipophilicityof musk compounds (logKow>4.6), sorption onto sludge could playan important role in their elimination from the liquid phase, whichwill be further discussed in the following section.

3.3. Behaviour of PPCPs in the bioreactor. Application of mass balances

The relative contribution of biological transformation, sorptionand volatilisation to the overall removal depends on the specific

(kbiol values in L g�1 d�1, calculated at warmer temperature and SRT 20–40 d) and the

Influencing parameters

Overall SRT T Rint

�� No No No�� No No No�� No No No++ No Yes Yes++ No No Yes++ No No Yes++ Yes Yes No+/++ Yes No No�+ No No Yes�+/+ Yes Yes n.a.+/++ No No n.a.�+/+ No No n.a.�+/+ No Yes n.a.++ No No n.a.++ No No n.a.�+/+ No No n.a.

tention time (SRT), temperature (T) and internal recirculation ratio (Rint) on the

stream (i)Feed Anox in Anox out Aer out Sed in Sed out Effluent

F CBZ

,i (µ

g d

-1)

0

1000

2000

3000

4000

(B)

stream (i)Feed Anox in Anox out Aer out Sed in Sed out Effluent

0

1000

2000

3000

4000

F CBZ

,i (µ

g d

-1)

(A)

Fig. 2. Total mass flow of CBZ in the different streams of the pilot plant working at colder (A) and warmer (B) temperatures and at SRT of <20 (j), 20–40 ( ) and >40 d ( ).

168 S. Suárez et al. / Chemosphere 89 (2012) 164–171

properties of each compound, mainly its chemical structure (reac-tivity of their moieties), Kd and H, respectively. Total flows werecalculated according to Eq. (3) for the different streams in theexperimental set-up (Fig. 1) and the removals in each compart-ment determined according to Eq. (4). Data were classified accord-ing to the operational temperature (colder: 14–18 and warmer:18–23 �C), SRT (<20, 20–40 and >40 d) and internal recirculationratio (Rint 3 or 4). Additionally kbiol was estimated according toEq. (6) for the selected PPCPs for the operation at the higher tem-perature and SRT of 20–40 d in order to perform a rough estima-tion of the biodegradability of the compounds (Table 1).

Regardless of the temperature, SRT or Rint considered, CBZ, DZPand DCF were the most recalcitrant compounds out of the selectedPPCPs. Fig. 2 shows the mass balances profiles for CBZ, which arerepresentative of the other two persistent substances.

The compounds FLX, CTL, NPX and IBP are not significantlysorbed onto sludge according to their low sorption coefficient

(B

0

250

500

750

F NXP

,i (µ

g d

-1)

(A)

(

stream (i)Feed Anox in Anox out Aer out Sed in Sed out Effluent

F FLX

,i (µ

g d

-1)

0

300

600

900

(A)

Fig. 3. Total mass flow of NPX and FLX in the different streams of the pilot plant working( ) and when working at Rint 3 ( ). (h) Mass flow of compound sorbed to sludge.

(logKd < 1.1), but these compounds exhibited from intermediateto high biological degradation in the pilot plant (60% for CTL, 79–89% for FLX, 81–96% for NPX, 80–99% for IBP). Only for FLX partof the transformation was already observed in the anoxic compart-ment of the plant (Fig. 3).

Both, the natural (E1 + E2) and synthetic (EE2) estrogens shouldbe bio-transformed to a significant extent according to their re-ported kbiol (Joss et al., 2006). In addition, their logKd is around2.5, which means that they are slightly lipophilic and will thereforebe partially sorbed onto sludge. In the current study, hormoneswere transformed principally in the aerobic tank of the pilot plant,being E1 + E2 almost completely transformed (>93%), while for EE2slightly lower removal rates were achieved (74–85%). The enrich-ment of activated sludge in nitrifying bacteria seems to positivelyinfluence the removal of EE2, probably through hydroxylation ofthe compounds (Vader et al., 2000; Forrez et al., 2008), which couldbe responsible for the high transformations measured in this work.

F NPX

,i (µ

g d

-1)

0

200

400

600)

stream (i)Feed Anox in Anox out Aer out Sed in Sed out Effluent

F FLX

,i (µ

g d

-1)

0

300

600

900

B)

at colder (A) and warmer (B) temperatures, at SRT of <20 (j), 20–40 ( ) and >40 d

S. Suárez et al. / Chemosphere 89 (2012) 164–171 169

According to the mass balances, around 70% of estrogens present inthe pilot plant were sorbed onto sludge which could have favouredtheir degradation.

Antibiotics (SMX, TMP, ROX and ERY) have similar sorptionbehaviour as estrogens, with the exception of ERY whose Kd isnegligible (Göbel et al., 2005), but significantly lower reported bio-degradation constants (Joss et al., 2006). Even so, appreciabletransformations have been observed for the four antibiotics (33–86%). Partial transformation under anoxic redox conditions hasonly been observed in the case of ERY and TMP. Biodegradabilityof SMX and TMP was determined by Perez et al. (2005), who re-ported 74% of SMX elimination within 3 d. On the contrary, TMPwas only found to be biodegraded by nitrifying sludge.

Fragrances illustrate the coexistence of the three removalmechanisms: volatilisation, sorption and biodegradation, althoughin the present study volatilisation was only significant for ADBI(Fig. 4). Fragrances were the only compounds for which Kd couldbe experimentally determined, obtaining the following result (inL kg�1): 2.7 ± 1.9 � 103 for HHCB, 1.0 ± 0.9 � 103 for AHTN and6.4 ± 4.0 � 102 for ADBI. These values are in the same order asthose obtained by Ternes et al. (2004a) for HHCB and AHTN,although almost one order of magnitude below the values reportedby Kupper et al. (2006) for the three compounds. The high contri-bution of sorption onto sludge on the total mass flow of musk com-pounds inside the pilot plant is clearly shown in Fig. 4, which isrelated to their strong lipophilic character. Transformation ofHHCB and AHNT during biological treatment reached 81–90%and 84–94%, respectively, being part of the removal already ob-served in the first anoxic compartment. The detection of themetabolite HHCB-lactone in Bester (2004) indicates that HHCBcan be biologically transformed, being the removals reported inprevious studies in the range of 16–50% (Bester, 2004; Joss et al.,2005; Kupper et al., 2006). Some previous works with AHTN indi-cate sorption as the only mechanism responsible for its removal(Bester, 2004; Joss et al., 2005), although Kupper et al. (2006) asso-ciated 43% of the depletion of AHTN to degradation. The overall

F HH

CB,

i (µ g

d -1

)

0

1500

3000

4500

(A) (

(

stream (i)Feed Anox in Anox out Aer out Sed in Sed out Effluent

F ADBI

,i (µg

L -1

)

0

2000

4000

6000(A)

Fig. 4. Total mass flow of HHCB and ADBI in the different streams of the pilot plant wor( ).Contribution of sorption (h) and volatilisation ( ) to the mass flow of these compo

removal of ADBI attained in the pilot plant was in the same rangeas for the other two fragrances considered (85–94%), althoughtransformation only accounted for 44–77%, being the rest removedby stripping.

3.4. Effects of operational parameters on the removal of PPCPs

Some operational parameters of the pilot plant, such as HRT,composition of the synthetic feed and dissolved oxygen level havebeen maintained constant during the whole process, whereas tem-perature, SRT and the internal recirculation flow varied. Accord-ingly, their influence on the process could be evaluated. Theeffect of temperature and SRT have been considered for all com-pounds, whereas the effect of the internal recirculation ratio couldonly be analysed for the first group of compounds spiked to thesystem.

3.4.1. Influence of SRTThe SRT of the plant exerted a significant effect only on the

transformation degree of compounds with a significant sorptionpotential such as estrogens and SMX. For lipophilic substancesthe retention time inside the reactor might be stronger influencedby the SRT than by the HRT of the plant, which could explain howcompounds with relatively slow kinetics can be biologically trans-formed during the secondary treatment step when operating athigh SRTs. In the case of musk fragrances, transformations ob-served in the pilot plant (80–95%) were considerably higher thanreported values (Bester, 2004; Joss et al., 2005; Kupper et al.,2006), which could be attributed to the higher SRT consideredfor this pilot plant compared to the cited data (for whichSRT < 25 d). In the case of natural estrogens the lowest elimination(93%) was observed at the lower SRT and temperature, which im-proved to >98% when one of these two parameters was raised,whereas EE2 removal increased 11% when working at SRT above20 d. The removal efficiency of SMX increased from 38 up to 63–70% when either SRT or temperature was increased.

F HH

CB,

i (µ g

d -1

)

0

1000

2000

3000

B)

stream (i)Feed Anox in Anox out Aer out Sed in Sed out Effluent

F AD

BI,i

(µg

L -1

)

0

750

1500

2250B)

king at colder (A) and warmer (B) temperatures and at SRT of 20–40 (j) and >40 dunds.

0

20

40

60

80

100

E1+E2 FLX ERY SMX CBZ

Rem

oval

(%)

Higher T

Lower T

kbiol: >300 ~10 ~1 <0.1 <0.01(L gSS d-1)

Fig. 5. Influence of temperature on the removal selected PPCPs which exhibitdifferent biodegradation constants (kbiol from Joss et al. (2006)). Adapted from Omilet al. (2010).

0

1

2

3

4

5

6

0

20

40

60

80

100

120

0 200 400 600 800 1000 1200

VSS

(g L

-1)

Rem

oval

effi

cien

cy (%

)'

time (d)

Fig. 6. Evolution in the removal of NPX in the pilot plant (�) compared to theconcentration of VSS inside the reactor (+). Adapted from Omil et al. (2010).

170 S. Suárez et al. / Chemosphere 89 (2012) 164–171

3.4.2. Influence of internal recirculation ratioThe increase in the internal recirculation ratio led to a slight

improvement (10%) in the removal efficiency of IBP, NPX andFLX, which were already transformed to a high extent (70–80%)at the first period of the operation with Rint of 3. In the case ofCTL the effect was stronger, since its removal increased from 25%to more than 50% when the internal recirculation ratio was set at4. More effective mixing in the reactor could partially be responsi-ble for that, although it could also be due to the higher oxygentransport from the aerobic to the anoxic compartment, in whichthe compounds could be to some extent aerobically transformed.This effect might be especially relevant for substances with moder-ate biological degradation constants, as has been observed in thepresent work for CTL.

3.4.3. Influence of redox conditionsOperating at different redox conditions might result in an in-

creased microbial diversity and a broader enzymatic spectrum in-side the bioreactor. In a previous work by Suarez et al. (2010) thefate of PPCPs was studied in lab-scale bioreactors working undernitrifying and denitrifying conditions. Those results pointed outthat, although biological degradation of PPCPs might occur at bothenvironments, some specific compounds (NPX, EE2, ROX and ERY)are only degraded under aerobic conditions. However, data avail-able for biological processes that combine different redox condi-tions within the same unit are scarce. In this research, thecalculated mass balances permit to identify where the removal ofeach one of the PPCPs is achieved at a higher extent, thus enablingthe possibility to get a deeper insight into the influence of redoxconditions in systems designed for nutrients removal. PPCPs withsignificant transformation rates during the anoxic process wereAHTN, HHCB, ADBI, FLX, TMP and ERY (Table 1), while the remain-ing PPCPs were only removed in the aerobic compartment. Formost PPCPs results are in agreement with those obtained by Suarezet al. (2010) except for natural estrogens and ERY. Some previousauthors reported the effect of redox conditions on the behaviourof estrogens. For example, Joss et al. (2004) observed in batchexperiments that degradation of E1 and E2 takes place underanaerobic, anoxic and aerobic environments, whereas EE2 is onlysignificantly removed under aerobic conditions and at slower ratesthan natural estrogens. On the other hand and similarly to the re-sults of this research, they observed that the match between modelcalculations and measured values in STPs improved if no degrada-tion of natural estrogens was assumed in the first anoxic reactordespite their degradation potential under those conditions, whichthey attributed to competitive inhibition of their degradation by

the influent substrate. The exposure to higher nitrate concentra-tions in the pure anoxic reactors compared to the pilot plant, couldalso explain this discrepancy. Concerning ERY, no significant an-oxic transformation would be expected from the results obtainedby Suarez et al. (2010), although as previously indicated the anoxiccompartment of the pilot plant is not totally free of oxygen due tothe high Rint applied, meaning that the transformation measuredfor ERY could be partially aerobic.

3.4.4. Influence of temperatureThe positive effect of warm temperature comparing to moder-

ate ones was particularly observed for E1 + E2, FLX, ERY andSMX, being the influence highest for the last compound (�30%)and lowest for the first one (�5%). This means that the influenceof temperature is inversely proportional to the biological degrada-tion rate constants of PPCPs (Fig. 5). Consequently, temperature is arelevant factor for substances with moderate to low kbiol thatundergo transformation following mechanisms which involvemicrobial activity.

3.4.5. Influence of sludge (concentration and acclimation)The influence of this parameter was observed for the anti-

inflammatory NPX. The performance of the pilot plant regardingNPX removal increased from 27% up to 99% during the first 300 d(Fig. 6), indicating a possible acclimation of bacteria to this phar-maceutical. Acclimation of biomass is known to be beneficial fordegradation of xenobiotics although its influence on the removalof PPCPs has only been reported in few cases. Ternes et al.(2004b) pointed out the possibility that existing microorganismscould acclimate to the presence of PPCPs by broadening their enzy-matic spectrum, in response to the lower sludge loading with bulkorganics when working at higher SRT. After day 600 a clear corre-lation between sludge concentration in the pilot plant and the effi-ciency in the elimination of NPX can be observed (Fig. 6). Thisconfirms the hypothesis that biological transformation of pharma-ceuticals follows pseudo-first order kinetics (Eq. (5)), with directproportionality of the transformation rate to the soluble substanceconcentration, as well as to the sludge concentration, although theeffect of the latter will only be significant for compounds withmoderate biological degradation constants.

4. Conclusions

High removal was observed for FLX, NPX and IBP, due to theirgood biodegradability. FLX and NPX were positively affected bytemperature and sludge concentration, respectively, which areoperation parameters that directly influence kinetics. Estrogenswere efficiently biodegraded, especially at the higher SRT due tomoderate sorption.

S. Suárez et al. / Chemosphere 89 (2012) 164–171 171

Fragrances were highly transformed already during the anoxicstage, presumably due to their enhanced retention inside the reac-tor. Volatilisation showed to be an important removal mechanismfor ADBI. Higher temperatures and SRT can lead to high efficienciesin biological removal of antibiotics.

For CTL the transformation degree in the pilot plant was around50%, when the reactor operation was optimised in terms of nitro-gen removal.

Acknowledgments

This work was supported by Spanish Ministry of Education andScience (INNOTRAZA project: CTQ2010-20240, NOVEDAR_Consol-ider project: CSD2007-00055) and Xunta de Galicia (GRC Program:2010/37). The authors thank Oliver Gans (Austrian Federal Envi-ronment Agency) for the analyses of antibiotics.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.chemosphere.2012.05.094.

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