Chemosphere 91 (2013) 1165–1175

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Chemosphere

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Use of effect-directed analysis for the identification of organic toxicantsin surface flow constructed wetland sediments

0045-6535/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.chemosphere.2013.01.023

⇑ Corresponding author. Tel.: +34 988368897.E-mail address: jorge.regueiro@uvigo.es (J. Regueiro).

Jorge Regueiro a,⇑, Víctor Matamoros b, Rémi Thibaut b, Cinta Porte b, Josep M. Bayona b

a Nutrition and Bromatology Group, Analytical and Food Chemistry Department, Faculty of Food Science and Technology, Ourense Campus, University of Vigo, E-32004 Ourense, Spainb Environmental Chemistry Department, IDAEA-CSIC, Jordi Girona 18-26, E-08034 Barcelona, Spain

h i g h l i g h t s

" Toxicity of constructed wetland sediments was assessed by effect-directed analysis." Cell viability and EROD induction in PLHC-1 cells were used as endpoints." A selective extract fractionation was performed by PLE and normal phase HPLC." Several PAHs, pharmaceuticals, musk fragrances and pesticides were found.

a r t i c l e i n f o

Article history:Received 25 May 2012Received in revised form 28 November 2012Accepted 2 January 2013Available online 8 February 2013

Keywords:Effect-directed analysisBioassay-directed fractionationToxicityConstructed wetlandOrganic microcontaminants

a b s t r a c t

Wetlands constitute one of the most efficient ecosystems with a great capacity to recycle the organicmatter and able to attenuate or mitigate the chemical pollution. However, limited information existson the ecotoxicological effects that may be caused due to the presence of these pollutants in wetland sed-iments. In this work, a bioassay-directed approach was used to identify toxicologically active compoundsretained in sediments from a surface flow constructed wetland located in the North-Eastern of Spain. Sed-iment fractionation was accomplished by pressurized-liquid extraction (PLE) followed by semiprepara-tive normal phase high performance liquid chromatography (NP-HPLC). During the extractionprocedure, different solvents were sequentially applied in order to selectively extract the compoundsas a function of their polarity. The cytotoxicity of the resulting fractions was assessed on the fish hepa-toma cell line PLHC-1 by using the thiazolyl blue tetrazolium bromide (MTT) assay, while the presence ofCYP1A inducing agents was determined by measuring the activity 7-ethoxyresorufin-O-deethylase(EROD) in exposed cells. Identification of the compounds was performed by gas chromatography coupledto mass spectrometry (GC/MS). Compounds such as polycyclic aromatic hydrocarbons (PAHs), non-ste-roidal anti-inflammatory drugs (NSAIDs), polycyclic musk fragrances and pesticides were identified inthe most toxic fractions.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

In the environment, chemicals occur as complex mixtures aris-ing from both anthropogenic and natural sources. Therefore, legis-lation is based on a list of priority pollutants but non-targetcomponents may also contribute to the overall sample toxicityand are neglected in the monitoring programmes (Brack, 2003).Thus, the identification of compounds responsible for toxicity inenvironmental samples is of great interest in order to update thepriority lists and to their further regulation.

Bearing this in mind, biological and analytical techniques havebeen combined to isolate and characterise the biologically active

compounds. The effect-directed analysis (EDA), also known as bio-assay-directed chemical analysis (Fernandez et al., 1992), aims toestablish a causal link between chemical substances and biologicaleffects in environmental samples (Brack, 2003; Grung et al., 2007).It is based on a concept known as ‘‘toxicity identification evalua-tion’’ that was introduced by the US Environmental ProtectionAgency (US EPA) in the 1980s (Mount and Anderson-Carnahan,1988; Norberg-King et al., 1991).

The basic idea of EDA is to reduce progressively the complexityof environmental samples by removing non-toxic components inorder to facilitate a chemical identification of the remaining toxi-cants. It involves the separation of sample extracts into coherentpieces, called fractions, which contain organic chemicals of similarfunctionality or polarity. Toxic potency of the fractions is thentested by applying bioassays and the obtained results are used to

1166 J. Regueiro et al. / Chemosphere 91 (2013) 1165–1175

focus the chemical analysis towards the most toxic fractions(Brack, 2003; Scheurell et al., 2007). This bioanalytical approachhas been demonstrated as a valuable tool for identifying toxicantsin various types of environmental matrices such as soils, sedi-ments, air particulate matter, effluents and groundwater (Grifollet al., 1992; Hannigan et al., 1998; Brack et al., 2005; Grunget al., 2007; Schmitt et al., 2011).

The selection of suitable toxicological bioassays is essential forthe identification of toxicants. Thus, in vitro cell-based bioassaysare simple, rapid, sensitive, cost-effective and require a smallamount of sample (Fent and Bätscher, 2000; Fernandes et al.,2002). Furthermore, many of these assays utilise the same end-points that are used in studies with whole organisms, which some-times provides a useful link between in vitro and in vivo effects(Knauer et al., 2007).

Urban and industrial wastewaters contain a variety of organiccontaminants such as pharmaceuticals and personal care products(PPCPs), pesticides and veterinary drugs. Some of them are onlypartially removed in conventional wastewater treatment plants(WWTPs). This issue can be overcome by the introduction of bio-logical cleaning systems such as constructed wetlands (CWs), astertiary treatment system. CWs are land-based wastewater treat-ment systems that use natural processes involving wetland vegeta-tion, substrates, and their associated microbial assemblages toimprove water quality (Fent and Bätscher, 2000). The main advan-tages of CWs are their low operational costs, the fact that they donot require an external energy source and their integration withthe landscape. Surface-flow constructed wetlands (SFCWs), usedas a tertiary treatment, have been reported to be useful for remov-ing a large variety of organic microcontaminants (Conkle et al.,2008; Matamoros et al., 2008; Matamoros and Salvadó, 2012). Nev-ertheless, the wetland sediment is a subject of particular interest,as it can act as a sink for contaminants associated with particles,especially for those presenting a higher lipophilicity.

The aim of the present study was to identify the major organictoxicants occurring in the sediment from a SFCW, which operatesas a tertiary treatment by using an EDA approach. The CW, whichwas constructed as a part of a series of environmental restorationactivities in a high density population area (e.g. Granollers, Barce-lona) in North-Eastern Spain, is fed with a secondary-treatedwastewater from a conventional WWTP. Regarding to the EDA ap-proach, pressurized-liquid extraction (PLE) was employed as anexhaustive extraction technique whereas extract fractionationwas carried out by semipreparative high-performance liquid chro-matography (HPLC). Then, sediment toxicity was assessed on thefish hepatoma cell line PLHC-1 by using two endpoints: (a) cyto-toxicity determined with the thiazolyl blue tetrazolium bromide(MTT) cell viability assay; and (b) the presence of CYP1A inducingagents in sediment extracts determined by measuring 7-ethoxy-resorufin-O-deethylase (EROD) activity in exposed cells (Thibautand Porte, 2008). Finally, the chemical characterization of theresulting active fractions by gas chromatography coupled to massspectrometry (GC/MS) allowed detecting several organic pollutantssuch as polycyclic aromatic hydrocarbons (PAHs), non-steroidalanti-inflammatory drugs (NSAIDs), polycyclic musk fragrancesand pesticides. Finally, the acute toxicity of the identified mole-cules was estimated by the Ecological Structure Activity Relation-ship (ECOSAR) modelling.

2. Materials and methods

2.1. Chemicals

HPLC grade methanol, acetonitrile, acetone, ethyl acetate,dichloromethane (DCM), n-hexane and trifluoroacetic acid (TFA)

were obtained from Merck (Darmstadt, Germany). Certified stan-dards PAH-Mix 9, containing 16 polycyclic aromatic hydrocarbons(acenaphthene, acenaphthylene, anthracene, benzo(a)anthracene,benzo(b)fluoranthene, benzo(k)fluoranthene, benzo(g,h,i)perylene,benzo(a)pyrene, chrysene, dibenz(a,h)anthracene, fluoranthene,fluorene, indeno(1,2,3,c,d)pyrene, naphthalene, phenanthrene, pyr-ene), and Pesticide-Mix 14, containing 16 organochlorine pesti-cides (aldrin, 4,40-DDD, 4,40-DDE, 2,40-DDT, 4,40-DDT, dieldrin,alpha-endosulfan, beta-endosulfan, endrin, alpha-HCH, beta-HCH,gamma-HCH, heptachlor, trans-heptachlor epoxide, hexachloro-benzene and methoxychlor), were supplied by Dr. Ehrenstorfer(Augsburg, Germany). The musk compounds celestolide (ADBI),phantolide (AHMI), galaxolide (HHCB), tonalide (AHTN) and tra-seolide (ATII) were obtained from Promochem Iberia (Barcelona,Spain). Cashmeran (DPMI) was kindly supplied by Ventos (Cornellade Llobregat, Barcelona, Spain). Triclosan, propyl-4-hydroxybenzo-ate (propylparaben), butyl-4-hydroxybenzoate (butylparaben),mecoprop, methyl chlorophenoxy acetic acid (MCPA), terbutyl-azine, clofibric acid, carbamazepine, methyl dihydrojasmonate,ibuprofen, naproxen, ketoprofen and diclofenac were purchasedfrom Sigma–Aldrich (Steinheim, Germany). Trimethylsulfoniumhydroxide (TMSH) was purchased from Fluka (Buchs, Switzerland).

Eagle’s Minimum Essential Medium, foetal bovine serum, L-glu-tamine, sodium pyruvate, nonessential amino acids, penicillin G,streptomycin, phosphate buffered saline (PBS) and trypsin–EDTAwere from Gibco BRL Life Technologies (Paisley, Scotland, UK).Thiazolyl blue tetrazolium bromide (MTT), 7-ethoxyresorufin, 7-hydroxyresorufin, and b-naphthoflavone (BNF) were purchasedfrom Sigma (Steinheim, Germany). For bioassay purposes, sedi-ment extracts were prepared in ethanol, whereas BNF was dis-solved in DMSO. Additions to the complete growth medium weremade so that the final solvent concentration never exceeded 0.4%(v/v), which was not cytotoxic.

2.2. Constructed wetland description and sampling

The SFCW is fed with a small fraction (0.4%) of the secondaryeffluent from the Granollers WWTP, which serves 154000 inhabit-ants. The WWTP influent flow rate consists of about 45% industrialwastewater and 55% domestic sewage with an average total flow of23700 m3 d�1. The WWTP carries out pre-treatment, primary clar-ification, activated sludge treatment and secondary clarification.

The SFCW is constituted by a single cell with a surface area of1 ha. It has shallow stretches of water planted with Typha sp. andPhragmites australis, unplanted deep stretches of water and a smallisland. These different areas were created in order to increase thepotential biodiversity of the system. The system started to operatein April 2003. The wetland treats approximately 100 m3 d�1 with ahydraulic retention time of around 1 month. Wetland sedimentsamples were randomly collected at a water depth of 1.5 m, corre-sponding to the unplanted zone. All samples were freeze-dried,homogenised, sieved through a 120 lm and stored at �20 �C untilanalysis.

2.3. Sample preparation

A pool of five wetland sediment samples was extracted using apressurized-liquid extraction device onePSE (Applied Separations,Allentown, PA, USA). Aliquots of 5 g of wetland sediment weretransferred to the extraction cell and then filled with previouslywashed sand. The extraction was carried out in an 11 mL cell at100 �C, 100 bar and 5 min static time (3 cycles). Three different sol-vents were sequentially used as extractants and the obtained ex-tracts were separately collected in 50 mL vessels. The selectedsolvents were: (a) 3% acetone in hexane (F1), (b) 0.02% trifluoro-acetic in acetone (F2), and (c) methanol (F3). Elemental sulphur

J. Regueiro et al. / Chemosphere 91 (2013) 1165–1175 1167

was removed by the addition of a small amount of activated copperand overnight stirring at room temperature. The recovered extractswere concentrated to a smaller volume (1 mL) by rotary evapora-tion and then evaporated to dryness under a gentle stream ofnitrogen.

In total, five wetland sediment subsamples (25 g) were ex-tracted to obtain enough amounts of extracts for the further frac-tionation and characterization. Dry extracts were gravimetricallydetermined and then separated into two portions, used for bioas-say testing and HPLC fractionation respectively.

2.4. HPLC fractionation

Extracts showing biological effect were fractionated by normal-phase HPLC (NP-HPLC) on a semipreparative aminopropyl silicacolumn Hypersil APS-2 (250 mm � 7 mm, particle size 10 lm)from Thermo Hypersil-Keystone (Bellefonte, PA, USA). Fraction-ation was performed using a dual pump Shimadzu LC-10A HPLCsystem (Shimadzu, Kyoto, Japan) in combination with a UV/VISdetector SPD-10A operating at 254 nm combined with a fractioncollector FRC-10A, also from Shimadzu. Injections were carriedout using a Rheodyne valve (Rohnert Park, CA) with a sample loopvolume of 500 lL fitted to a Shimadzu autosampler SIL-10AD. TheF1 extract was reconstituted in hexane and separated into threefractions at equal time intervals using a gradient elution programat a flow rate of 2 mL min�1 in 40 min. Hexane was pumped iso-cratically during 10 min, followed by a linear gradient from 100%hexane to 100% DCM over 15 min and held at 100% DCM for15 min. The F2 extract was reconstituted in hexane/ethyl acetate(3:1) and 4 fractions were collected at 10 min intervals at a flowrate of 2 mL min�1 over 40 min. The selected gradient elutionwas hexane/ethyl acetate (3:1) isocratically pumped during10 min, followed by a linear gradient up to 100% DCM over15 min and held at 100% DCM during 15 min. HPLC fractions wereconcentrated to 1 mL using a rotary evaporator and then evapo-rated to dryness under a gentle stream of nitrogen. Dry weightswere gravimetrically determined and every extract was split intotwo portions. One of them was employed for bioassay testing,whereas the other was reconstituted in 1 mL of ethyl acetate andused for chemical identification by GC/MS. Fractions were labelledas Fi.j, where i indicates the fraction number in the PLE extractionand j the fraction number in the HPLC fractionation.

2.5. Bioassay testing

2.5.1. Cell cultureThe PLHC-1 cells derived from a hepatocellular carcinoma in

topminnow (Poeciliopsis lucida) were obtained from ATCC (CRL-2406, passage 88). They were grown in Eagle’s Minimum EssentialMedium supplemented with 5% foetal bovine serum, 2 mM L-gluta-mine, 1 mM sodium pyruvate, 0.1 mM nonessential amino acids,1.5 g L�1 sodium bicarbonate and 50 U mL�1 penicillin G/50 lg mL�1 streptomycin in a humidified incubator with 5% CO2

at 30 �C. Cells were routinely cultured in 75 cm2 polystyrene flaskswhere 2.5 � 106 cells were seeded. When 90% confluence wasreached, approximately after 7 d, cells were dissociated with0.05% (w/v) trypsin and 0.5 mM EDTA for subculturing and exper-iments. Experiments were carried out on confluent cell monolayersand on passages 3–30.

2.5.2. MTT cell viability assayCells were seeded at a rate of 75000–150000 cells per well

(depending on the duration of the exposure) in 96-well platesand allowed to attach for 24 h in a humidified incubator with 5%CO2 at 30 �C. The medium was then changed and replaced by med-ium containing the wetland sediment extracts (1, 2.5 and

5 lg mL�1) or ethanol (0.4% carrier solvent). Plates were returnedto the incubator for 6 or 24 h of incubation. Treatments were car-ried out in quintuplicate.

After chemical exposure the medium was aspired, the cellswere rinsed with 100 lL of PBS and immediately incubated with0.5 mg mL�1 MTT dissolved in complete growth medium. Plateswere further incubated for 2 h. MTT solution was removed and200 lL DMSO/ethanol (1:1) was added and allowed to dissolvethe intracellular formazan crystals for 1 h in the dark. Absorptionwas measured at 570 nm on a microplate reader (Varioskan, Ther-mo Electron Corporation). Cell viability was expressed as a per-centage of the controls (cells exposed to the solvent).

2.5.3. Induction of 7-ethoxyresorufin-O-deethylase activitySediment extract fractions were reconstituted in ethanol and

the EROD activity was used as a marker of the presence of CYP1Ainducing agents. Cells were exposed for 6 h to the extract fractions.Cells were seeded at a rate of 650000 cells per well (500 lL) in 48-well plates and allowed to grow for 48 h in a humidified incubatorwith 5% CO2 at 30 �C. The medium was then changed and replacedby medium containing BNF (positive control), sediment extracts orcarrier solvents. Treatments were carried out in triplicates andplates were returned to the incubator for the exposure period.After chemical exposure, the medium was aspired and the cellswere rinsed with 500 lL of PBS. Then, cells were immediately incu-bated with 2 lM of 7-ethoxyresorufin in 50 mM Na-phosphatebuffer pH 8.0 at 30 �C. After 15 min of incubation in the microplatereader (Varioskan, Thermo Electron Corporation), the fluorescencewas directly read at the excitation/emission wavelength pairs of537 and 583 nm. Quantification was made by calibration with 7-hydroxyresorufin.

EROD activity of each extract was compared to the induction re-sponse of the positive control BNF, which was included in eachplate to allow normalisation and comparison of results from differ-ent experiments. Data was expressed as potency index, and calcu-lated as: [(induction response of the tested extract � inductionresponse of the blank)/(induction response of the positive con-trol � induction response of the blank)] � 100.

2.6. Gas chromatography–mass spectrometry (GC/MS)

The chemical analyses were performed using a TRACE GC/MSsystem (Thermo-Finnigan, Dreieich, Germany) operated by Xcali-bur v1.2 data software. Separation was carried out on a DB-5 cap-illary column (30 m � 0.25 mm, 0.25 lm film thickness) from J&WScientific (Folsom, CA). Helium (99.9995% purity) was employed ascarrier gas at a constant linear average velocity of 40 cm s�1. TheGC oven temperature was programmed from 65 �C (held 2 min)to 120 �C at 15 �C min�1, at 4 �C min�1 to 160 �C, at 7 �C min�1 to220 �C, at 5 �C min�1 to 290 �C and finally at 15 �C min�1 to320 �C, holding the final temperature for 5 min (total analysistime: 55 min). 2 lL of each fraction were injected in a splitlessinjector at 270 �C with an injector purge activation time of0.8 min. In situ derivatisation of carboxylic acids was performedin the GC injector port by adding 10 lL of TMSH solution (0.25 Min methanol) to a 50 lL aliquot of each subfraction.

The quadrupole mass spectrometer was operated in the elec-tron impact (EI) ionisation positive mode (+70 eV). Instrumentalparameters for ionisation were as follows: filament emission cur-rent 150 lA, filament/multiplier delay 4 min, detector voltage550 V and perfluorotributylamine (PFTBA) as calibration gas. Themass range was scanned in the full scan mode from 50 to 500 m/z. The scan time for data acquisition was set at 0.5 s. Ion sourceand transfer line temperatures were maintained at 200 �C and280 �C respectively.

1168 J. Regueiro et al. / Chemosphere 91 (2013) 1165–1175

2.7. Data analysis

Experimental data were expressed as means ± standard devia-tion of three (EROD activity induction) and five replicates (cytotox-icity). Statistical analyses were performed using Student’s t-testwith the level of significance set at p 6 0.05. Aquatic toxicity pre-dictions were made using the US EPA’s ECOSAR model version1.1 (United States Environmental Protection Agency, Washington,DC, USA). Mass spectra deconvolution was performed using theAutomated Mass Spectral Deconvolution and Identification System(AMDIS) version 2.68 (National Institute of Standards and Technol-ogy, Gaithersburg, MD, USA).

3. Results and discussion

3.1. PLE fractionation

Several solvents (isooctane, hexane, ethyl acetate, acetone, ace-tonitrile, methanol and ethanol) and combinations were initiallyevaluated in order to achieve a suitable PLE fractionation. The com-binations evaluated (10% ethyl acetate in hexane, 3% acetone inhexane, 20% acetone in methanol and 50% methanol in acetoni-trile) were selected on the base of previous experience in fraction-ation procedures, aiming to cover a wide range of polarities. Theresulting extracts were analysed by HPLC–UV/VIS in order to selectthose showing the highest responses.

The best fractionation of the wetland sediment was obtainedwith the following sequence of extractants: (F1) 3% acetone in hex-ane, (F2) 0.02% trifluoroacetic acid in acetone, and (F3) methanol(Fig. 1). The resulting extracts were evaporated to dryness andthe corresponding residues were gravimetrically determined. Themost polar fraction (F3) showed the highest mass weight extracted(68%), which indicated an important presence of polar compoundsincluding humic acids in the wetland sediment. The least polarfraction (F1) accounted for 22% of the total, whereas F2 only pre-sented 10% of the total content.

All three PLE extracts were subjected to MTT cell viability andEROD induction assays in order to evaluate their toxicity and thepresence of CYP1A inducing agents in the extract. MTT assay isbased on the absorption by cells of thiazolyl blue tetrazolium bro-mide and its reduction by a mitochondrial dehydrogenase to darkblue formazan crystals which are impermeable to cell membranes,thus resulting in its accumulation within the healthy cells. Thecontent of the formazan created is directly proportional to the

F1

F1.1 F1.2 F1.3

NP-HPLC(APS-2)

Wetland sedime

PLE 13% acetone in hexa

PLE 20.02% TFA in aceto

pH=2

PLE 3MeOHF3

Fig. 1. Sample preparation scheme for the effect-

number of surviving cells and, therefore this assay can be used asa measure of cytotoxicity. As shown in Fig. 2a, only the least polarextract F1 presented a significant cytotoxic effect in cell culturesexposed to 5 lg mL�1. Thus, the cell viability was of 70% after 6 hof exposure, whereas it decreased to 48% after 24 h.

The EROD assay involves the oxidative deethylation of the sub-strate 7-ethoxyresorufin to fluorescent resorufin by CYP1A, an iso-enzyme of cytochrome P-450 that is induced by a variety oftoxicants, including planar PAHs and halogenated aromatic com-pounds, non-steroidal anti-inflammatory drugs, fibrates, anti-depressives, etc. (Thibaut and Porte, 2008). Therefore, this assayprovides evidence of exposure to chemicals that exhibit dioxin-likeproperties. Maximal induction of EROD activity was reached after6 h of exposure to 1 lM BNF (100% potency index), and corre-sponded to 24-fold induction over the activity of the blank (cellsexposed only to the carrier solvent). As shown in Fig. 2b, sedimentextracts induced EROD activity in a dose–response manner. At alltested concentration levels, the highest activity was observed forthe least polar extract F1, with a potency index comprised between6% and 25%. The F2 extract also significantly induced EROD activity,exhibiting a potency index of 18% when tested at 5 lg mL�1. Themethanolic extract F3 showed no significant EROD activity evenat the higher level of exposure, which indicated the absence ofCYP1A inducers in this fraction.

In spite of being the fraction containing the highest amount ofmass extracted (70%), the methanolic extract (F3) was the onlyone not active in both assays at the studied concentrations. There-fore, this fraction was not further considered in the present study.This result suggests that compounds responsible for the observedtoxicity in the wetland sediment possess medium-to-low polari-ties. Low concentrations of polar toxicants are expected to occurin the sediments due to the high lipophilicity of this kind of matrix.Bearing this in mind, it is plausible that SFCW water may also showtoxicity, although further studies are needed to check thispossibility.

3.2. HPLC fractionation

To reduce their complexity, those PLE extracts showing a rele-vant activity in MTT or EROD assays were fractionated by semipre-parative HPLC. Taking into account the apolar nature of F1 and F2extracts, the normal-phase mode was selected as the most suitableoption for their fractionation. F1 was separated into three subfrac-tions with increasing polarity (F1.1, F1.2 and F1.3), whereas four

nt

ne

ne F2

F2.1 F2.2 F2.3 F2.4

NP-HPLC(APS-2)

directed fractionation of wetland sediments.

Fig. 2. (a) Time-dependent cytotoxicity of the PLE sediment extracts at a concen-tration of 5 lg mL�1 in PLHC-1 cells. �Significantly different from control (p < 0.05).Values are means ± standard deviation (n = 5). (b) Dose–response induction ofEROD activity in PLHC-1 cells after 6 h of incubation with different concentrationsof the PLE sediment extracts. �Significantly different from control (p < 0.05). Valuesare means ± standard deviation (n = 3).

J. Regueiro et al. / Chemosphere 91 (2013) 1165–1175 1169

subfractions were collected from F2, namely F2.1, F2.2, F2.3 andF2.4 (Fig. 3).

Amongst the F1 subfractions, the most polar one (F1.3) ac-counted for the highest mass (52%), whereas F1.2 contained

40

60

80

100 Absorbance at 254 nm

0

20

F1.1 F1.2

% D

ichl

orom

etha

ne

0 5 10 15 20

Absorbance at 254 nm

40

60

80

100

F2.1 F2.2

0

20

0 5 10 15 20

% D

ichl

orom

etha

ne

Fig. 3. HPLC chromatograms of the normal-phas

approximately a 27% and F1.1 only a 7%. Regarding the F2 subfrac-tions, F2.1 and F2.3 presented the highest mass percentages with a33% and a 27%, respectively. F2.4 accounted for the 14% of F2 mass,whereas F2.2 only contained a 9%. A 14% of F1 and a 17% of F2 masswere not recovered, probably due to losses during the HPLC frac-tionation procedure.

The MTT cell viability and the EROD induction assays were con-ducted on all collected subfractions in order to establish which ofthem displayed the highest activities. A statistically significantcytotoxic effect was observed in cell cultures exposed to 5 lg mL�1

of F1.3 (Fig. 4a). After 6 h of exposure to this subfraction, the cellviability accounted for 60%, and after 24 h of exposure it decreasedto 51%. No significant effect could be observed for the rest of theextracts, except for F2.2, which induce a slight increase in cell via-bility (112 ± 3%) after 24 h of exposure. When tested at 5 lg mL�1,all the extracts induced EROD activity in a dose–response manner,with a potency index comprised from 10% to 36% (Fig. 4b). Thehighest inductions (P20%) were observed for F1.2, F2.1 and F2.2.These subfractions were still active at a concentration of2.5 lg mL�1, with potency indices over 10%. Subfraction F1.3showed an EROD activity below 10% at all tested concentrations,although this value might be underestimated due to the reducedcell viability observed for this subfraction.

In summary, the EROD activities induced by the crude PLE ex-tracts were significantly lower than the sum of the correspondingsubfractions. This finding may suggest antagonic effects of interfer-ing compounds that were removed by NP-HPLC fractionation.Through decreasing the complexity of environmental samples byfractionation, the probability of such antagonic effects also de-creases. Similar antagonic effects were already detected in dis-solved and particulate matter of a polluted river (Grifoll et al.,1992) and sediments (Fernandez et al., 1992).

3.3. Toxicants identification

The HPLC subfractions showing highest toxicological activitieswere characterised by GC/MS in an attempt to identify candidatetoxicants in each subfraction (Table 1). Only peaks with a signal-

40

20

0

Time (min)

F1.3100

80

60

% H

exane

0

25 30 35 40

F2.3 F2.4

60

40

20

Time (min)25 30 35 40

100

80

% H

exane/AcOEt (3:1)

e fractionation of the PLE sediment extracts.

Fig. 4. (a) Time-dependent cytotoxicity of the different subfractions of the wetland sediment extracts at a concentration of 5 lg mL�1 in PLHC-1 cells. �Significantly differentfrom control (p < 0.05). Values are means ± standard deviation (n = 5). (b) Dose–response induction of EROD activity in PLHC-1 cells after 6 h of incubation with the differentsubfractions of the wetland sediment extracts. �Significantly different from control (p < 0.05). Values are means ± standard deviation (n = 3).

1170 J. Regueiro et al. / Chemosphere 91 (2013) 1165–1175

to-noise ratio above 20 were considered for identification. The ac-quired mass spectra were deconvoluted and then compared to ref-erence spectra in the 2008 version of the NIST/EPA/NIH MassSpectral Library (NIST 08) for tentative identification. Most of thethus identified compounds were then positively identified by com-parison of their mass spectra and retention times to those of avail-able standard solutions, although they were not quantitativelydetermined in the present study.

Metabolic inhibition, which was assessed by the MTT assay, isnormally observed for a mixture of different compounds, and can-not easily be attributed to one or a few compounds. Nevertheless, avariety of environmental organic pollutants such as pesticides andpharmaceuticals are known to produce cytotoxicity in vitro (Fentand Hunn, 1996; Laville et al., 2004; Knauer et al., 2007).

Several compounds such as pharmaceuticals (e.g. diclofenacand ibuprofen), biocides (triclosan), PAHs (phenanthrene), musks(cashmeran) and surfactants (octylphenol) with octanol/water par-tition coefficients (logKow) ranging from 3.97 to 4.90, were identi-fied in the most cytotoxic subfraction F1.3. These findings are inquite good agreement with the toxicological effects shown forthese compounds in fish and Daphnia estimated by using the ECO-SAR model (Table 1). This program uses Structure Activity Rela-tionships (SARs), mainly based on the octanol–water partitioncoefficient, to estimate the median lethal dose (LC50). Amongstthe compounds identified in the cytotoxic subfraction F1.3, 4-tert-octylphenol, triclosan and methyltriclosan showed the highestpredicted toxicities with LC50 values below 0.5 mg L�1. Further-more, the MTT cytotoxicity of the non-steroidal anti-inflammatorydrugs (NSAIDs) diclofenac and ibuprofen has been previously re-ported in different fish cell cultures, showing half maximal effec-tive concentration (EC50) values ranging from 0.019 to 1.20 mMfor the PLHC-1 line (Laville et al., 2004; Caminada et al., 2006; Thi-baut and Porte, 2008). Diclofenac seems to be the compound

exhibiting the highest acute toxicity within the class of NSAIDs(Fent et al., 2006) and it is commonly found in wastewater at themicrogram per gram level (Ternes, 1998; Matamoros et al.,2008). The biocide triclosan is widely used as an antibacterialand antifungal agent in various domestic products, and it is oftenfound at high concentrations in wastewater and sludge (Bester,2005; Heidler and Halden, 2007). Triclosan has adverse effects onaquatic organisms, being highly toxic to algae and to some speciesof fish, particularly in their early stages of development (Orvoset al., 2002; Dann and Hontela, 2011). The polycyclic aromatichydrocarbon (PAH) phenanthrene, the polycyclic musk cashmeran,4-tert-octylphenol and piperonyl butoxide were also identified inthis subfraction F1.3. Piperonyl butoxide is a synergist used to en-hance the effectiveness of pesticides, whereas 4-tert-octylphenol isthe degradation intermediate of the widely employed industrialnon-ionic surfactants. Although the cytotoxicity of several of thesecompounds has been reported to be relatively low when testedseparately in PLHC-1 cells (Fent and Hunn, 1996; Choi and Oris,2000; Fent and Bätscher, 2000), the observed effect may suggestthe existence of synergisms. Thus for example, the presence ofpiperonyl butoxide has been reported to increase the MTT cytotox-icity of fibrates, anti-inflammatory drugs and fluvoxamine inPLHC-1 cell cultures (Thibaut and Porte, 2008).

The subfraction F1.2 presented the highest EROD response butwas not cytotoxic for the PLHC-1 cells. Most of the identified com-pounds in this subfraction belonged to the group of polycyclicmusks (i.e. galaxolide, celestolide, phantolide and tonalide), PAHs(fluoranthene and pyrene), antioxidants (2,4,6-tri-tertbutylphenoland butylated hydroxytoluene) and UV filters (octyl methoxycin-namate). As can be observed in Table 1, these compounds pre-sented the lowest predicted LC50 values in fish (96-h) andDaphnia (48-h), which disagrees with the low cytotoxicity ob-served for this subfraction (Fig. 4). It should be noted, however,

Table 1Compounds identified in the different subfractions of the wetland sediment.

Fraction Compound CASnumber

LogKow Fish 96-hLC50a

(mg L�1)

Daphnia 48-hLC50a (mg L�1)

Planarcompound

Structure

F1.2 2,4,6-Tri-tertbutylphenol 732-26-3

6.06 0.024 0.045 +

F1.2 2,6-Diisopropylnaphthalene

24157-81-1

5.80 0.042 0.043 ++

F1.2 Acetyl cedrene 32388-55-9

5.60 0.528 0.439 �

F1.2 Amberonne 54464-57-2

5.18 0.285 0.257 �

F1.2 Butylatedhydroxytoluene

128-37-0

5.10 0.199 0.221 +

F1.2 Celestolide 13171-00-1

6.60 0.066 0.066 +

F1.2 Fluoranthene 206-44-0

5.16 0.409 0.356 ++++

F1.2 Galaxolide 1222-05-5

5.90 0.036 0.038 +

F1.2 Octyl methoxycinnamate 5466-77-3

5.80 0.136 0.124 +

F1.2 Phantolide 15323-35-0

6.70 0.077 0.076 +

F1.2 Pyrene 129-00-0

4.90 0.409 0.356 ++++

(continued on next page)

J. Regueiro et al. / Chemosphere 91 (2013) 1165–1175 1171

Table 1 (continued)

Fraction Compound CASnumber

LogKow Fish 96-hLC50a

(mg L�1)

Daphnia 48-hLC50a (mg L�1)

Planarcompound

Structure

F1.2 Tonalide 1506-02-1

5.70 0.030 0.032 +

F1.2 Traseolide 68140-48-7

6.30 0.032 0.034 +

F1.3 4-Tert-octylphenol 140-66-9

4.12 0.123 0.150 +

F1.3 Phenanthrene 85-01-8

4.34 1.187 0.947 +++

F1.3 Cashmeran 33704-61-9

4.9 1.241 0.968 �

F1.3 Diclofenac 15307-86-5

4.51 38.442 29.202 +

F1.3 Ibuprofen 15687-27-1

3.97 42.036 30.899 +

F1.3 Methyltriclosan 4640-01-1

4.90 0.341 0.309 +

F1.3 Piperonyl butoxide 51-03-6

4.75 3.005 2.287 +

F1.3 Triclosan 3380-34-5

4.76 0.484 0.469 +

F2.1 Anthraquinone 84-65-1

3.40 10.531 7.244 +++

F2.1 Benzophenone 119-61-9

3.18 13.746 9.184 +

F2.1 Butylparaben 94-26-8

3.50 2.429 1.492 +

1172 J. Regueiro et al. / Chemosphere 91 (2013) 1165–1175

Table 1 (continued)

Fraction Compound CASnumber

LogKow Fish 96-hLC50a

(mg L�1)

Daphnia 48-hLC50a (mg L�1)

Planarcompound

Structure

F2.1 Methyl chlorophenoxyacetic acid

94-74-6

3.25 539.179 328.335 +

F2.1 Naproxen 22204-53-1

3.18 189.971 126.094 ++

F2.1 Tertbuthylazine 5915-41-3

3.21 2.799 4.615 +

F2.2 Carbamazepine 298-46-4

2.50 44.672 76.247 +++

F2.2 Clofibric acid 882-09-7

2.84 299.345 191.225 +

F2.2 Ketoprofen 22071-15-4

3.17 258.367 168.906 +

F2.2 Mecoprop 7085-19-0

3.13 247.535 160.336 +

F2.2 Methyldihydrojasmonate 24851-98-7

2.7 8.525 15.453 �

F2.2 Propylparaben 94-13-3

2.94 5.157 2.627 +

F2.2 Tris(2-chloroethyl)phosphate

115-96-8

1.40 62.865 135.463 �

F2.2 Tris(2-chloroisopropyl)phosphate

13674-84-5

2.60 13.864 25.419 �

a Estimated by ECOSAR model (US EPA, 2012).

J. Regueiro et al. / Chemosphere 91 (2013) 1165–1175 1173

that ECOSAR approach generally is well suited for chemicals thatexert their toxicity through general membrane disruption, butmay be problematic for chemicals for which the mode of toxic ac-tion is uncertain or highly specific (Moore et al., 2003). Galaxolideand tonalide are extensively used as fragrances in a large range ofhousehold products including detergents, soaps and personal-care

products (PCPs) (Rimkus, 1999; Bester, 2009). These compoundshave been detected in sewage sludge, WWTP effluents and surfacewaters impacted by WWTPs (Gatermann et al., 2002). Although lit-tle is known about the ability to induce EROD activity of this kindof chemical compounds, tonalide has been reported to slightly in-crease EROD activity in PLHC-1 cell line at concentrations between

1174 J. Regueiro et al. / Chemosphere 91 (2013) 1165–1175

2 and 4 lM (Torre et al., 2011). PAHs such as fluoranthene and pyr-ene, and an industrial chemical (2,6-diisopropylnaphthalene), allwith planar polycyclic structures (Table 1), were found in this sub-fraction. Although no significant EROD induction was observedwith fluoranthene in PLHC-1 cells, a concentration-related induc-tion of CYP1A activity was obtained with pyrene (Fent and Bät-scher, 2000). Traven et al. (2008) also found, on a general scale,strong correlation between CYP1A induction and the concentrationof PAHs in several contaminated marine samples. The antioxidantcompounds such as 2,4,6-tri-tertbutylphenol and butylatedhydroxytoluene detected in this fraction are mainly used as addi-tives in cosmetics, pharmaceuticals, jet fuels, rubber and petro-leum products (Kolpin et al., 2002). UV filters such as octylmethoxycinnamate could be also identified in this subfraction.Due to its extensive use in cosmetics and sunscreen products, thiscompound can be present in wastewaters and sludge at high con-centrations (Negreira et al., 2011a,b).

Subfractions F2.1 and F2.2 which also induced EROD activity,contained pharmaceuticals, herbicides, organophosphate fire retar-dants and fragrances with a logKow ranging from 1.4 to 3.4. Theidentification of several pharmaceuticals such as naproxen, car-bamazepine, clofibric acid and ketoprofen in these subfractions isin agreement with the high concentration of these compoundsfound in the aquatic environment (Ternes, 1998; Fent et al.,2006). Among them, clofibrate and naproxen were found to bemoderate inducers of EROD activity in PLHC-1 cells, whereas dic-lofenac (present in subfraction F1.3) and ketoprofen were veryweak inducers (Thibaut and Porte, 2008). Furthermore, the herbi-cides terbuthylazine, mecoprop and MCPA were detected in thesesubfractions (Table 1). Other identified compounds included pre-servative additives such as butylparaben and propylparaben, usu-ally found in wastewaters (Regueiro et al., 2009), as well asorganophosphate flame retardants such as tri(2-chloroethyl) phos-phate and tris(2-chloroisopropyl) phosphate, and also the fra-grance methyldihydrojasmonate, widely used in householdproducts. The presence of these pharmaceutical, herbicide and per-sonal care products in the wetland-sediment was in agreementwith the high abundance of these substances previously found inwater samples collected from the SFCW (Matamoros et al., 2008).

4. Conclusions

An effect-directed approach was used to trace and identify tox-icologically active compounds in a wetland sediment from a SFCWin North-Eastern Spain. A primary in-cell fractionation by PLE wascarried out using several solvents mixtures with different polari-ties. MTT cell viability and EROD induction in the fish hepatomacell line PLHC-1 were selected as toxicological endpoints. The ac-tive fractions were further fractionated by NP-HPLC into fine frac-tions. The highest activities were observed for fractions obtainedfrom extractions with 3% acetone in hexane and 0.02% trifluoroace-tic acid in acetone, suggesting a low polarity of the present toxi-cants. Several compounds could be identified by GC/MS,including PAHs, NSAIDs, polycyclic musk fragrances and pesticides.ECOSAR model approach was employed in an attempt to predictin vivo acute aquatic toxicity of the identified compounds, althoughobtained results were not fully correlated with the observed cyto-toxicity in vitro.

Although many compounds were identified as possible toxi-cants in the analysed fractions, many others remained unknown,the use of high resolution mass spectrometry detectors and theassessment of potential interactions such as synergism or antago-nism among contaminants present in the samples at environmen-tal concentrations are need to get a further insight on theenvironmental identification of toxicological active compounds.

Acknowledgements

This research was supported by the projects CTM2008-06676-C05-04/TECNO from Spanish Ministry of Science and Innovationand 2009SGR924 from Generalitat de Catalunya. J.R. would liketo acknowledge the Ministerio de Ciencia e Innovacion of Spainfor his Juan de la Cierva contract.

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