Activities and identification of aryl hydrocarbon receptor agonists in sediments from the Danube...

ORIGINAL PAPER

Activities and identification of aryl hydrocarbon receptoragonists in sediments from the Danube river

Steffen Keiter & Stefanie Grund & Bert van Bavel &Jessika Hagberg & Magnus Engwall &Ulrike Kammann & Martin Klempt & Werner Manz &

Helena Olsman & Thomas Braunbeck & Henner Hollert

Received: 31 July 2007 /Revised: 21 September 2007 /Accepted: 25 September 2007 /Published online: 16 October 2007# Springer-Verlag 2007

Abstract This study is a consequence of a distinct fishdecline in the Danube river since the beginning of the1990s. In contrast to the decline of fish population, formerstudies have repeatedly documented that the water qualityalong the Danube river is improving. However, the conclu-sion of a pilot study in 2002 was that a high hazard potential

is associated with local sediments. The present study docu-ments that sediment samples from the Danube river showedcomparatively high aryl hydrocarbon receptor mediatedactivity in biotests, using the cell lines GPC.2D.Luc, H4IIE(DR-CALUX®) and RTL-W1. The combination of chemicalanalysis, fractionation techniques and different in vitro testsrevealed that priority pollutants could not explain the maininduction, even though the concentrations of prioritypolycyclic aromatic hydrocarbons (PAHs) were very high(maximum in the tributary Schwarzach, sum of 16 EPAPAHs 26 μg/g). In conclusion, this investigation shows thatnonpriority pollutants mainly mediate the high inductionrates. Nevertheless, owing to the effects of PAHs towardsfish and the connection between dioxin-like activity andcarcinogenicity, the link between contamination and the fishpopulation decline cannot be ruled out.

Keywords Danube . Fish decline . Dioxin-like activity .

Aryl hydrocarbon receptor agonists .Weight of evidence

Introduction

Over the last two decades, a decline of fish catches hasbeen reported for several streams in Europe, USA andCanada [1–5]. Particularly in the upper Danube river be-tween Sigmaringen and Ulm, the fish populations havedecreased markedly since the beginning of the 1990s [5, 6].Despite continuous stocking programs, Wurm [6] reportedsignificant declines in the numbers of various fish species,in particular grayling (Thymallus thymallus). However,sewage purification and water quality along the Danuberiver were improved, as could repeatedly be documented byconventional chemical analysis of some priority pollutantsand monitoring of different hydrological and biological

Anal Bioanal Chem (2008) 390:2009–2019DOI 10.1007/s00216-007-1652-x

S. Keiter (*) : S. Grund : T. Braunbeck :H. HollertAquatic Ecology and Toxicology Section, Department of Zoology,University of Heidelberg,Im Neuenheimer Feld 230,69120 Heidelberg, Germanye-mail: [email protected]

B. van Bavel : J. Hagberg :M. Engwall :H. OlsmanDepartment of Natural Science, Man-Technology-EnvironmentResearch Centre (MTM), University of Örebro,70182 Örebro, Sweden

U. KammannInstitute for Fishery Ecology,Federal Research Centre for Fisheries,Palmaille 9,22769 Hamburg, Germany

M. KlemptDepartment of Fish Quality,Federal Research Centre for Nutrition and Food,Palmaille 9,22769 Hamburg, Germany

W. ManzFederal Institute of Hydrology,Am Mainzer Tor 1,56068 Koblenz, Germany

H. HollertDepartment for Ecosystem Analyses, Institute for EnvironmentalResearch (Biology V), RWTH Aachen University,Worringerweg 1,52074 Aachen, Germany

parameters [7]. The main objective of a pilot investigationin 2002 [5] was to assess the ecotoxicological hazardpotential in the upper Danube river using one line ofevidence: biological effect monitoring. The results of thisstudy clearly showed that acute and specific toxic potentialsare associated with local sediments, suspended particulatematter and effluents. The conclusion was that morecomprehensive weight-of-evidence studies using additionallines of evidence [8] are needed to elucidate if the declineof fish population is caused by chemicals or by other,nontoxic parameters. Furthermore, small tributaries shouldbe integrated as potential sources of additional contamina-tion. The present investigation is one part of this compre-hensive weight-of-evidence framework and was designed todetermine by means of biological and chemical analyses ifcontamination by priority persistent organic pollutants(POPs) with dioxin-like activity can be associated withlocal sediments from the upper Danube river and selectedtributaries.

Halogenated aromatic hydrocarbons (HAHs) and poly-cyclic aromatic hydrocarbons (PAHs) are chemicals usuallyrelated to dioxin-like activity [9]. In water, these chemicalsare intimately associated with suspended particles andconsequently with sediments. Both groups of organicchemicals are known to act as aryl hydrocarbon receptor(AhR) agonists [10–13]. In cells these chemicals bind to theAhR, and the ligand–receptor complex undergoes atransformation process during its translocation into thenucleus. There it binds to the dioxin responsive element,which is the promoter of the cytochrome P4501A (CYP1A)and other AhR-regulated genes [14]. As a consequence, thetranscription of these genes is activated, an event assumedto be an initial step in the onset of most of the biochemicaland toxic effects observed upon exposure to dioxin-likechemicals [14, 15].

HAH contamination of the environment mostly involvescomplexmixtures of polychlorinated dibenzodioxins (PCDDs),polychlorinated dibenzofurans (PCDFs), polychlorinated bi-phenyls (PCBs) and other HAHs with a wide spectrum ofAhR binding affinities [10]. HAHs induce a number of toxicresponses in vertebrates, including hepatotoxicity, bodyweight loss, thymic atrophy, impairment of immune re-sponses, reproductive toxicity and thyroid metabolism,teratogenicity and carcinogenesis [16–19]. Although thereare 419 possible PCDD, PCDF and PCB congeners, only21 are known to be highly toxic to fish [2], especiallyagainst early life stages of fish [19–21]. Typical toxicresponses are malformations, hemorrhages and formation ofpericardial edema [19, 22].

Several studies have shown that not only HAHs, but alsoPAHs can act as AhR agonists [23]. Furthermore, a majorportion of AhR-mediated dioxin-like activity in sedimentswas caused by nonpriority PAHs [24, 25]. Brack et al. [25]

demonstrated that especially PAHs with a molecular weightbetween 228 and 252 explain the major dioxin-likepotencies of sediment extracts from the Neckar river basin(Germany). The principal effects currently associated withexposure to PAHs are carcinogenicity, immunosuppressionand endocrine dysfunction [15, 23, 26–31]. D’Adamo et al.[32] showed that PAHs do not significantly bioaccumulatein fish because of highly efficient and rapid detoxificationmechanisms in the liver. However, several PAHs are wellknown to be mutagenic after bioactivation in the liver [26,29]. Bioactivation of PAHs is primarily mediated bycytochrome P450 and epoxide hydrolase.

Owing to the lack of knowledge about the presence oforganic priority pollutants in sediments of the upper Danuberiver and its tributaries, one objective of the present studywas to quantify concentrations of PAHs, PCBs and PCDDs/PCDFs, to obtain an overview of the hazard potential of localsediments. Furthermore, regarding the fish population de-cline in the upper Danube river and the results of previousstudies [5], it seemed important to investigate the dioxin-like activity of sediment samples. As already demonstratedin the literature, chronic exposure to different HAHs andPAHs may affect genetic diversity and, therefore, reducethe average fitness of organisms at the population level [3,33]. Thus, a fish cell line (RTL-W1 rainbow trout liverfibroblasts) as well as two different mammalian cell lines(recombinant H4IIE rat hepatoma cells and GPC.2D.Lucguinea pig adenocarcinoma cells) were chosen to measurethe relative potency of sediment extracts expressed as2,3,7,8-TCDD (TCDD) equivalent concentrations. Thedifferent test systems were compared with respect to theirsuitability to demonstrate potential links between bioassayresults, chemical analyses and, eventually, the fish popula-tion decline in the upper Danube river. Additionally, TCDDequivalent concentrations (chemical toxicity equivalents,Chem-TEQs) [17, 34–37] of selected organic substanceswere determined and related to the induction of dioxin-likeactivities in the bioassays. In particular, by the combinationof chemical and biological analysis it is possible tocalculate the percentage of unknown nonpriority pollutantsin the sediments and, therefore, to compensate for thedisadvantages of both techniques.

Materials and methods

Sediment samples

Near-surface sediment samples (0–5-cm depth) werecollected with a van Veen gripper or directly with glassbottles from eight sites along the Danube river and fromtwo tributaries (Fig. 1). Immediately after return to thelaboratory, sediment samples were frozen at −30 °C and

2010 Anal Bioanal Chem (2008) 390:2009–2019

freeze-dried using a Christ Alpha 1–4 freeze dryer (Christ,Osterode, Germany). Amounts of 20 g of the dried sed-iment samples were separately extracted with acetone(Fluka, Buchs, Switzerland) for 12 h using standard reflux(Soxhlet) extractors at approximately ten cycles per hour[38]. The extracts were reduced in volume to approximately5 mL using a rotatory evaporator (WB 2001; Heidolph,Kehlheim, Germany; 400 mbar, 36–38 °C). Under anitrogen stream, extracts were reduced close to dryness.Residues from each sample were redissolved in 1 mLdimethyl sulfoxide (DMSO; Fluka) for bioassays and in1 mL toluene (Fluka) for chemical analysis of PCBs andPCDDs/PCDFs respectively (Fig. 2). Extracts were storedat −20 °C until testing. Empty extraction thimbles weresubjected to extraction and processed in two parallelexperiments to serve as process controls.

Multilayer fractionation

In order to provide information on the identities ofunknown substances inducing dioxin-like activities inwhole extracts, a multilayer fractionation was performedto remove acid-degradable compounds (nonpersistent, e.g.,PAHs). Samples were cleaned on an open “sandwich” silicacolumn (mesh 15 mm; Merck, Darmstadt, Germany)

packed with KOH silica (30 mm), neutral silica (5 mm),40% H2SO4 silica (30 mm), 20% H2SO4 silica (15 mm),neutral silica (10 mm) and Na2SO4 (10 mm), and wereeluted with n-hexane (Riedel-de Haen, Seelze, Germany).The remaining fraction contained persistent dioxin-likecompounds and included, e.g., PCDDs/PCDFs and PCBs;some PAHs, however, are known not to be retained in themultilayer fraction [39]. The solvents were divided in twoequal portions and the first parts were evaporated under anitrogen stream, and the sample was transferred to DMSO(Fluka) for subsequent bioassays (Fig. 2).

To separate planar and nonplanar compounds, the secondparts of the samples were fractionated using an open carboncolumn (Carbopack C; Fluka). Adsorbent (1.5 g), carbon onCelite (1:9; Celite 545, 20–45 μm, Fluka) and a layer ofNa2SO4 were packed in glass columns. The sample waseluted into two subsequent fractions with 10 mL of hexane(nonplanar compounds) and 80 mL of toluene (coplanarcompounds). After the addition of a recovery standard (13C-labeled 1,2,3,4-TCDD, 1,2,3,7,8,9-HxCDD), the sampleswere evaporated and transferred to amber glass autosamplervials in 25 μL of tetradecane. The extracts and standardswere stored at −18 °C until high-resolution gas chromatog-raphy (HRGC)/high-resolution mass spectrometry (HRMS)analysis.

Freeze-dried sediments

Soxhlet: crude extracts

PAH analysis

Bioassay

Silica multilayer column (MF)

Bioassay

Carbon column

PCDD/F analysis

PCB analysis Fig. 2 Conceptual frameworkon the biological and chemicalanalysis applied in this study.PAH polycyclic aromatic hydro-carbon, PCB polychlorinatedbiphenyl, PCDD/F polychlori-nated dibenzodioxin and diben-zofuran, MF multilayer fraction

Fig. 1 Sampling sites alongthe Danube river betweenSigmaringen and Jochenstein. 1Sigmaringen, 2 Lauchert(tributary), 3 Riedlingen, 4Schwarzach (tributary), 5Rottenacker, 6 Ehingen, 7Oepfingen, 8 Ingolstadt, 9 BadAbach, 10 Jochenstein

Anal Bioanal Chem (2008) 390:2009–2019 2011

PCBs and PCDDs/PCDFs—HRGC/HRMS analysis

HRGC/HRMS analysis was performed with a MicromassAutospec Ultima instrument operating at more than10,000–12,000 resolution using electron impact ionizationat 35 eV. All measurements were performed in the selectiveion recording mode, monitoring the two most abundant ionsin the chlorine cluster. Splitless injection of 1 μL of thefinal extract was used on a 60-m Rtx dioxin 2 column(0.25-mm inner diameter, 25 μm). In addition to a blanksample with each set of samples (five to ten), a laboratoryreference material was analyzed.

PAH analysis

Chemical analyses were carried out according to standardmethods described elsewhere [40]. Briefly, the determina-tion of 16 PAHs (naphthalene, acenaphthylene, acenaph-thene, fluorene, phenanthrene, anthracene, fluoranthene,pyrene, benzo[a]anthracene, chrysene, benzo[b,j]fluoran-thene, benzo[k]fluoranthene, benzo[a]pyrene, dibenzo[a,h]anthracene, benzo[ghi]perylene, indeno [1,2,3-cd]pyrene;US EPA 610) was carried out by high-pressure liquidchromatography (gradient elution) with fluorescence anddiode-array detection according to DIN 38414-S21.

Bioassays

Test systems were selected to give a broad overview ofpotential molecular reactions to dioxin-like compounds,taking into account both cell-specific and species-specificdifferences (Table 1).

DR-CALUX assay

The DR-CALUX assay utilizes a rat hepatoma cell linewith a luciferase reporter gene controlled by the AhR(Biodetection Systems, Amsterdam, Netherlands) [22].DR-CALUX cells were seeded in 96-well plates (TPP,Trasadingen, Switzerland) in aliquots of 100 μL, 24 h priorto exposure under standard conditions of 37 °C and 5%CO2, and allowed to attain 100% confluence. Thereafter,sample dilutions were prepared in culture medium supple-

mented with 10% fetal calf serum (FCS), and added to thecells in triplicate wells. The extracts were tested in tenconcentrations in 3× dilution series, with a maximum con-centration of 2 mg/mL. In each assay, a calibration curve ofTCDD (0–300 pM) was tested. The final concentration ofDMSO (Fluka) was 0.8 or 1%, and the total volume was200 μL per well. After 24 h of exposure, the medium wasremoved and the cells were washed twice with phosphate-buffered saline (PBS) and lysed in PBS at −20 °Covernight. After the substrate luciferin had been added,followed by incubation for approximately 1 h in darkness at20 °C, the activity of luciferase was measured using theLuclite assay kit (PerkinElmer, Upplands Väsby, Sweden).Cell lysates were transferred to a white 96-well plate (TPP)and luminescence was determined in a multiwell-platereader (Wallace 1420, Victor2; PerkinElmer, USA). Dose–response curves were analyzed with the program GraphPadPrism 4® (GraphPad, San Diego, USA), using the Hillequation. The bottom value was set to the response of thesolvent controls. The luciferase-inducing potency of thesamples was converted to toxic equivalents (TEQs) byrelating the EC25 value of each extract to the EC25 ofTCDD as described in [41] using the fixed effect levelquantification method [42]. Briefly, the sample concentra-tion that induced luciferase to 25% of the TCDD-inducedmaximum luciferase activity was designated as theEC25TCDD for that sample. The TEQ concentrations werecalculated as

Bio�TEQs pg=gð Þ ¼ TCDDEC25 pg=mLð Þextract EC25TCDD g=mLð Þ :

GPC.2D assay

The guinea pig GPC.2D.Luc liver cells were provided by T.A. Gasiewicz (University of Rochester, New York, USA)and cultivated in Dulbecco’s modified Eagle’s mediumcontaining 10% FCS and 2 mM glutamine in 75-cm2

culture flasks at 37 °C and 5% CO2 [43]. Cells weretrypsinized with trypsin/EDTA and transferred to 96-wellplates (25,000 cells per well; TPP). The cells were allowedto grow overnight. Subsequently, cells were exposed to aseries (0.0014–80 nM, 0.0042–25 pg/mL) of TCDD

Table 1 Characteristics of cell lines used in this study

Cell name Original cell Construct Dioxin responsive elements References

GPC.2D.Luc GPC.16 (guinea pig) p2DLuc 2 DREs in an artificial constructed element [55]H4L1.1c4 (DR-CALUX®)

H4IIE (rat) PGudLuc1.1 4 DREs in a 480-bp promoter region of the mouseCYP1A

[22, 54]

RTL-W1 RTL-W1 (rainbowtrout)

– 7-Ethoxyresorufin-O-deethylase [44]

DRE dioxin responsive element, CYP1A cytochrome P4501A


(Ehrenstorfer, Augsburg, Germany) or sediment extracts(DMSO concentration 0.6%) for 24 h. The maximumconcentration of the extracts was 20 g/mL DMSO. Allsamples were tested in triplicates of five different concen-trations. Every experiment was repeated independently atleast three times. After incubation, cells were washed withPBS and lysed in 50 μL lysis buffer (25 mmol glycogly-cerine, 1% Triton X-100, 10% glycerin). Luminescencemeasurements were performed using a FLUOstar Optimaplate reader (BMG Labtech, Offenburg, Germany) beforeand after injection of 30 μL of substrate [Tris(hydroxy-methyl)aminomethane (20 mM, pH 7.8), MgCl2 (5 mM),EDTA (0.1 mM), dithiotreitol (33 nM), coenzyme A(270 μM), luciferin (470 μM) and ATP (530 μM)]. Datawere calculated by subtracting the arithmetic mean for 5 sbefore injection of the substrate from the mean for 13 safter substrate injection. The data were analyzed using theprogram GraphPad Prism 4. EC25 values were calculatedfrom log-transformed data using a sigmoidal curve-fit withvariable slopes. The results were expressed relative toTCDD as biological toxicity equivalents (Bio-TEQs). Themean EC25 of TCDD was 0.04700 pmol/mL (15 pg/mL), ascalculated from a total of 35 experiments.

7-Ethoxyresorufin-O-deethylase assay

Induction of 7-ethoxyresorufin-O-deethylase (EROD) wasmeasured in the CYP1A-expressing permanent fish livercell line RTL-W1 (Oncorhynchus mykiss) [44] with slightmodifications to the method described by Behrens et al.[45] according to the protocol given recently by Seiler et al.[13]. RTL-W1 cells [44] were kindly provided by Niels C.Bols and Lucy Lee (University of Waterloo, Canada) andmaintained at 20 °C in 75-cm2 plastic culture flasks (TPP)without additional gassing in Leibovitz medium supple-mented with 8% FCS (Sigma, Deisenhofen, Germany), 1%penicillin/streptomycin and 1% neomycin sulfate (Sigma)[46]. Before exposure to the standards, cells were seeded in96-well plates (TPP) and allowed to grow to 100%confluence for 72 h. Subsequently, the medium wasremoved and the cells were exposed for 72 h to thestandards diluted in medium to give a final concentrationrange between 5×105 and 2.5×105 pg/mL test mediumusing eight dilutions with three replicates each (DMSOcontent per well less than 1%). TCDD (Promochem, Wesel,Germany) was serially diluted to give a final concentrationrange of 3.13–100 pM on two separate rows of each plateas a positive control. Induction was terminated by removingthe growth medium and freezing at −70 °C to lyse the cells.The deethylation of exogenous 7-ethoxyresorufin wasinitiated by adding 7-ethoxyresorufin to each well andincubating in the dark at room temperature for 10 minbefore addition of NADPH in PBS. The plates were

incubated for further 10 min and the reaction was stoppedby adding fluorescamine dissolved in acetonitrile. ERODactivity was measured fluorometrically after another 15 minusing a GENios plate reader (Tecan, Crailsheim, Germany;excitation 544 nm, emission 590 nm). Protein was deter-mined fluorometrically using the fluorescamine method(excitation 355 nm, emission 590 nm) [24, 47]. The con-centration–response curves for EROD induction in theRTL-W1 bioassay were computed by nonlinear regression(GraphPad Prism 4) using the classic sigmoid curve orBoltzmann curve as model equations [24]. The calculationof TEQs was done as described for the DR-CALUX assay.

Results

Bioassays

Dioxin-like activity of the crude sediment extracts

The three cell lines displayed differential sensitivity to thesediment extracts. All extracts caused a concentration-dependent increase in dioxin-like activity. Compared withother experimental series in different central European riversystems, Bio-TEQs of the sediment samples were relativelyhigh [48]. The dose–response curves varied in shape and inthe maximum induction level (data not shown). However,in most cases, dose–response curves were biphasic, mostlikely indicating the onset of cytotoxicity at higher sampleconcentrations.

The lowest induction rates for dioxin-like activity(expressed as TCDD equivalent concentrations) for mostsamples were obtained in the GPC.2D assay (Fig. 3). Thelowest Bio-TEQ values in each bioassay were measured forthe sediment extracts from Riedlingen and the tributaryLauchert. The highest inductions in almost any assay werefound for the samples from the Schwarzach, Rottenackerand Oepfingen. The remaining samples exhibited onlyslight conformity in the different bioassays. There was nocorrelation between the results of the GPC.2D and the DR-CALUX assays (Spearman rank order correlation coefficientr=0.21), as well as for the results of the GPC.2D andEROD assays (r=0.04). In contrast, a strong correlationwas found between the Bio-TEQs of the EROD and theDR-CALUX assays (r=0.84).

Dioxin-like activity of the multilayer fraction

Table 2 summarizes the dioxin-like activities of the mul-tilayer fractions (containing acid-resistant organic pollutantslike PCBs and PCDDs/PCDFs) of the sediment samplescollected from the upper Danube river. In the EROD assay,induction rates of dioxin-like activities in fractionated sam-


ples were all considerably lower than in the DR-CALUXassay. However, a slight correlation could be shown for theBio-TEQs of the EROD and the DR-CALUX assays (r=0.6). The multilayer fractions of the samples were not testedin the GPC.2D assay. All samples except those fromRiedlingen clearly induced dioxin-like activity. In eithertest, the lowest Bio-TEQs were determined for the samplefrom the tributary Lauchert (112 and 20 pg/g in the ERODand the DR-CALUX assays, respectively) and from theDanube location Ehingen (117 and 40 pg/g, respectively).

The strongest AhR response in the DR-CALUX assaywas recorded for the fraction derived from the Oepfingensediment sample (1,400 pg/g). Elevated dioxin-like ac-tivities could also be associated with the samples from

the Schwarzach (876 pg/g), Sigmaringen (633 pg/g) andRottenacker (210 pg/g).

The highest EROD induction was measured in the mul-tilayer fraction from the tributary Schwarzach (123 pg/g).Slightly lower Bio-TEQ values were determined forsamples from Oepfingen (70 pg/g), Sigmaringen (54 pg/g)and Rottenacker (44 pg/g). In comparison with crudeextracts, the multilayer fractions in both test systems alwaysshowed distinctly lower induction. Only the multilayerfraction from Sigmaringen showed a comparatively highinduction in the DR-CALUX assay. In this sampleapproximately 60% of the overall activity in the crudesediment extract was caused by acid-resistant compounds(data not shown).

Table 2 Dioxin-like activity of the multilayer fractions in the DR-CALUX and ethoxyresorufin-O-deethylase (EROD) assays (biological toxicityequivalents, Bio-TEQ) and the chemical toxicity equivalents (Chem-TEQ) of persistent organic pollutants [sum of polychlorinated biphenyl (PCB)and polychlorinated dibenzodioxin (PCDD)/polychlorinated dibenzofuran (PCDF) toxic equivalents), as well as the percentage of the measuredchemicals responsible for the biological activity

EROD DR-CALUX

Bio-TEQ (pg/g) Chem-TEQ (pg/g) Percentage Bio-TEQ (pg/g) Chem-TEQ (pg/g) Percentage

Sigmaringen 54 83 153 633 35 6Lauchert 20 61 308 112 28 25Schwarzach 123 250 203 876 117 13Rottenacker 44 137 310 210 59 28Ehingen 40 110 273 117 47 40Oepfingen 63 303 478 1,400 126 9

Table 2 Dioxin-like activity of the multilayer fractions in the DR-CALUX and ethoxyresorufin-O-deethylase (EROD) assays (biologicaltoxicity equivalents, Bio-TEQ) and the chemical toxicity equivalents(Chem-TEQ) of persistent organic pollutants [sum of polychlorinated

biphenyl (PCB) and polychlorinated dibenzodioxin (PCDD)/poly-chlorinated dibenzofuran (PCDF) toxic equivalents), as well as thepercentage of the measured chemicals responsible for the biologicalactivity

Fig. 3 Dioxin-like activities of sediment extracts of the upper Danuberiver, as determined in the DR-CALUX® (H4IIE cells; n=1–3),GPC.2D (GPC.2D.Luc cells; n=3–6) and 7-ethoxyresorufin-O-dee-thylase (EROD; RTL-W1 cells; n=3) assays. Biological toxicity

equivalent (Bio-TEQ) values were calculated as the concentrationgiving 25% of the maximum induction of 2,3,7,8-TCDD. Data arepresented as mean±standard deviation


Chemical analysis: PAHs, PCBs and PCDDs/PCDFs

The results of chemical analysis of sediments from the Danuberiver are summarized in Tables 3 and 4. The highest con-centrations of the PCBs 77, 126 and 169 were found in sed-iments collected from the tributary Schwarzach (243 pg/g).PCB concentrations in sediments fromRottenacker (160 pg/g),Ehingen (92 pg/g) and Oepfingen (70 pg/g) were lower.The lowest PCB concentrations were measured for thesamples from Sigmaringen (37 pg/g) and the tributaryLauchert (23 pg/g).

Sediments from the tributary Schwarzach (5,328 pg/g)and the lake at Oepfingen (5,308 pg/g) showed the highestPCDD/PCDF concentrations. Lower concentrations weremeasured for the samples from Rottenacker (1,986 pg/g)and Ehingen (1,375 pg/g). The samples from the Lauchert

(1,269 pg/g) and Sigmaringen (1,428 pg/g) showed similarconcentrations.

The cumulative concentration of 16 analyzed PAHs (USEPA 610) of the sample from the tributary Schwarzach(26.3 μg/g) exhibits an approximately 5 times higher PAHlevel than the sediment sample from Oepfingen (5.3 μg/g).Similarly PAH concentrations were measured for thelocations Sigmaringen (2.0 μg/g), Ehingen (2.1 μg/g) andIngolstadt (1.4 μg/g). PAH concentrations under or slightlyabove 1 μg/g could be determined for the samples fromJochenstein (0.3 μg/g), the Lauchert (0.8 μg/g) and BadAbach (1.0 μg/g). The highest concentrations were mea-sured for fluoranthene (0.05–4.8 μg/g), phenanthrene(0.05–4.6 μg/g) and pyrene (0.1–3.4 μg/g). For Riedlingen,concentrations for each of the 16 PAHs measured wereconsistently below detection limits.

Combination of bioassays and chemical analyses

The results of chemical analyses were expressed as TCDDequivalent concentrations (Chem-TEQs), as calculated bymultiplying the concentration of each chemical with theavailable relative potency factor for the EROD (Fig. 4) andthe DR-CALUX (Fig. 5) assay [17, 34, 35]. Furthermore,for each sampling location the sum of the Chem-TEQs forthe different groups of chemicals was calculated [49]. Toelucidate the percentage of the measured chemicals (PAHs,PCBs and PCDDs/PCDFs) which is responsible for theinduction in the EROD and DR-CALUX assay when testingcrude extracts, the Chem-TEQs were subtracted from the

Table 4 Concentrations of the 16 US EPA polycyclic aromatic hydrocarbons (PAH) in sediment samples from the upper Danube river expressedas micrograms per gram of sediment

Sigmaringen Lauchert Schwarzach Ehingen Oepfingen Ingolstadt Bad Abach Jochenstein

Fluoranthene 0.38 0.14 4.8 0.40 0.95 0.25 0.17 0.05Pyrene 0.27 0.10 3.4 0.30 0.71 0.16 0.11 NDPhenathrene 0.15 0.05 4.6 0.19 0.38 0.11 0.11 0.02Benzo[a]pyrene 0.19 0.07 1.7 0.19 0.49 0.15 0.08 0.03Chrysene 0.18 0.07 1.9 0.18 0.44 0.14 0.12 0.03Benzo[b,j]fluoranthene 0.15 0.08 1.6 0.15 0.55 0.17 0.12 0.03Benzo[a]anthracene 0.18 0.06 2.2 0.18 0.43 0.10 0.07 NDIndeno[1,2,3-cd]pyrene 0.12 0.05 1.4 0.11 0.28 0.11 0.05 0.02Dibenzo[a,h]anthracene 0.16 0.07 0.49 0.15 0.39 0.02 0.04 NDBenzo[ghi]perylene 0.04 ND 0.97 0.04 0.10 0.11 0.1 0.04Benzo[k]fluoranthene 0.09 0.04 0.9 0.09 0.24 0.06 0.04 0.01Anthracene 0.07 0.03 1.0 0.07 0.19 0.03 0.02 0.01Fluorene ND ND 0.64 ND 0.04 ND ND NDAcenaphthylene 0.04 0.02 0.10 0.03 0.09 ND ND NDAcenaphthene ND ND 0.53 ND ND ND ND NDNaphthalene ND ND 0.09 ND 0.02 ND ND NDSum PAH (EPA) 2.03 0.79 26.32 2.08 5.30 1.41 1.02 0.24

ND not detectable (below detection limits)

Table 3 Sum concentrations of PCBs 77, 126 and 169 and PCDDs/PCDFs in sediment extracts from the Danube river expressed aspicograms per gram of sediment

PCDDs PCDFs PCBs

Sigmaringen 1,039.1 388.5 36.9Lauchert 1,102.6 166.3 23.2Schwarzach 4,807.5 520.1 243.0Riedlingen ND ND NDRottenacker 1,644.5 341.5 159.9Ehingen 1,109.5 265.6 92.3Oepfingen 4,589.1 719.1 70.4

ND. not detectable (below detection limits)


corresponding Bio-TEQs. For GPC.2D.DLuc cells, relativepotency factors for PAHs, PCBs and PCDDs/PCDFs arecurrently not available in the literature. Thus, TEQ valuescould not be calculated for GPC.2D.Luc cells.

For the majority of crude extracts from both the DR-CALUX and the EROD assay (Figs. 4, 5) less than 25% ofthe induction could be explained by known chemicalsexpressed as Chem-TEQs. For the tributary Schwarzachapproximately 50% of the induction could be explained bychemical analyses for both bioassays. Comparison of Bio-TEQs und Chem-TEQs calculated for the sample fromSigmaringen demonstrated that 55% of the induction isunknown using the DR-CALUX assay. However, especiallythe PAHs showed in each sediment sample the highestChem-TEQ values and, therefore, they are the most importantmeasured contributors of dioxin-like activity, even though themajority of the induction in both bioassays was caused byunknown compounds.

The induction measured in the EROD assay caused bythe multilayer fraction containing PCBs and PCDDs/PCDFs was at a comparatively low level. The percentageof the measured PCBs and PCDDs/PCDFs responsible forthe induction in this bioassay was over 100% for eachsample site (Table 2). In contrast, the percentage of themeasured PCBs and PCDDs/PCDFs in the multilayerfraction responsible for the dioxin-like activity in the DR-CALUX assay was not higher than approximately 40%(Ehingen). The highest response for the multilayer fractionin the DR-CALUX assay was calculated for the sample

fromOepfingen (1,400 pg/g), but only 9% could be explainedby measured concentrations of PCBs and PCDDs/PCDFs.

In almost any sample, the major portion of dioxin-likeactivity was caused by unidentified nonpriority and nonper-sistent pollutants in both the EROD and the DR-CALUXassay. In contrast, the majority of the dioxin-like activity ofcrude extracts from the Schwarzach could be explained bypriority PAHs measured chemically. A comparison of Figs. 4and 5 also documents that the multilayer fraction tested inthe DR-CALUX assay has a higher portion on the inductionof crude extracts than the EROD assay.

Discussion

All sediments from the Danube river tested inducedAhR-mediated activities in three dioxin-specific bioassays(DR-CALUX, GPC.2D and EROD assays), however withassay-specific differences. H4IIE cells showed slightlyhigher induction rates than RTL-W1 cells; both cell lines,however, gave significantly higher induction rates thanGPC.2D cells (H-test followed by post hoc test accordingto Dunn: p<0.05; data not shown). These observations canbe corroborated by means of correlation analyses: Nocorrelation was found between the results for the GPC.2Dcell line and the two other cell lines. In contrast, a distinctcorrelation could be obtained for the results with H4IIEand RTL-W1 cells (r=0.84). The moderate difference ininduction rates between these two cell lines might be ex-

Fig. 4 Comparison of the totalof the biological response in theEROD assay (Bio-TEQs), thecalculated contribution ofthe measured PAH , PCB andPCDD/PCDF toxic equivalents(TEQs) as well as the unknownportion (in percent) of the over-all activities regarding the Bio-TEQs of the crude extracts.*Substances below detectionlimits. **PAH or ***PCB aswell as PCDD/PCDF concen-trations are not available. PAH,PCB and PCDD/PCDF TEQswere calculated using the rela-tive potency factors taken fromBols et al. [34], Clemons et al.[35] and Safe [9], respectively


plained by species-specific induction mechanisms (Table 1)[50, 51], different exposure times as well as apparentsubstrate inhibition [22, 50–53].

These two hypotheses—EROD inhibition and longerexposure time in the RTL-W1 assay [22, 52]—would alsoexplain the comparatively low Bio-TEQ values in the mul-tilayer fractions using the EROD assay (Table 2). The Bio-TEQs determined by means of this assay were always belowthe calculated TEQs of PCBs and PCDDs/PCDFs concen-trations. Although it has been suggested that the metabolismrate for certain halogenated compounds is low owing tothe molecular size and steric hindrances [53], it is wellconceivable that the fish cell line degrades the substanceswithin the 72 h of incubation [51]. In contrast, the Bio-TEQsof the multilayer fractions in the DR-CALUX assay werealways above the TEQs of measured PCBs and PCDDs/PCDFs. In this case, the difference between Bio-TEQ andTEQ concentrations could be explained by other POPs whichhave not been investigated in chemical analyses.

The GPC.2D assay showed a 100-fold lower inductionthan the two other bioassays. Reasons for this lowinduction might be differences in the transfected responsiveelements as well as cell-specific and therefore chemical-dependent trans-acting factors and receptors [54, 55].Owing to this cell-specific induction, for the GPC cell linethere is a need to determine (1) the induction of severalpriority pollutants as single substances as well as mixtures,because it is well known that several POPs antagonize eachother in a species-specific manner, and (2) relative potency

factors to calculate TEQs for several single substances(PAHs, PCBs and PCDDs/PCDFs) for better comparabilitywith results of other cell lines.

In the case of concentration additivity [35, 49] in all crudesediment extracts, only a minor portion of the total potencyshown by the bioassays resulted from PCBs and PCDDs/PCDFs, as demonstrated by calculating the Chem-TEQs.The concentrations of these POPs responsible for the effectsdetermined in the DR-CALUX and the EROD assay werealways clearly below 8%. A distinctly higher inductionpotency in the different bioassays resulted from PAHs. Inthe sediment extract from the tributary Schwarzach, eventhe majority of the induction could be explained by con-centrations of priority PAHs (49.5 and 54.4% in the ERODand the DR-CALUX assay, respectively). However, for allother samples only a minor portion of the high dioxin-likeactivities of crude extracts could be explained by the priorityPAHs analyzed. In addition, the results of the multilayerfractions (containing only acid-resistant POPs) documentlow induction rates using the EROD assay. This confirms theresults of the chemical analysis, where POPs showed com-paratively low concentrations. Nevertheless, the dioxin-likeactivity of the multilayer fractions using the DR-CALUXassay always demonstrated relatively higher induction ratesof crude extracts than did the EROD assay. Thus, the DR-CALUX assay may be assumed to be more sensitive forPOPs than the EROD assay.

The aim of the chemical analysis was to obtain anoverview of concentrations of organic pollutants in sedi-

Fig. 5 Comparison of the totalof the biological response in theDR-CALUX assay (Bio-TEQs),the calculated contribution ofthe measured PAH, PCB andPCDD/PCDF TEQs as well asthe unknown portion (in per-cent) of the overall activitiesregarding the Bio-TEQs of thecrude extracts. *Substances be-low detection limits. **PAH or***PCB as well as PCDD/PCDF concentrations are notavailable. PAH, PCB andPCDDPCD/F TEQs were calcu-lated using the relative potencyfactors taken from Bols et al.[34], Clemons et al. [35] andSafe [9], respectively


ments from the upper Danube river. Therefore, well-investigated priority pollutants were chosen, which are alsowell known to induce dioxin-like activity. These inves-tigations demonstrated that especially for the small tributarySchwarzach and the lake at Oepfingen PAH concentrationswere on a comparatively high level (26.3 and 5.3 μg/g).According to the ATV classification scheme (ARGE-Elbecited in [24]) these two sample sites would be assigned toquality classes V and III, not reaching the quality goalsgiven by the ATV scheme for the highly contaminated Elberiver. Nevertheless, the origin of these high PAH concen-trations is still not identified. In contrast, the concentrationsof PCBs and PCDDs/PCDFs are comparatively low and,therefore, they would reach ATV quality class I (ARGE-Elbe cited in [24]). However, the highest concentration forthese substances were measured for sediments from thetributary Schwarzach and the lake at Oepfingen too (sum ofselected PCBs and PCDDs/PCDFs approximately 5.5 ng/g).Under consideration of the results from the bioassay, thesesample sites seem to be highly polluted hot spots of the upperDanube river.

Several other studies have shown that nonpriority pollu-tants may induceAhR-mediated activities and have a potentialcontribution to environmental hazards [24, 25]. For example,more polar compound classes such as heterocyclic aromatichydrocarbons, amines and several pesticides are also wellknown as AhR agonists [56]. In addition, a number oftypical natural compounds such as wood components, reteneand juvabione as well as various bioflavonoids have beenidentified as AhR ligands [57–59]. To summarize, the highdioxin-like activities of crude sediment extracts observed inthe cell lines RTL-W1 and H4IIE could not be explainedcompletely by priority PAHs nor by POPs. Therefore, weassume that the main induction in the biotest systems wascaused by unknown nonpriority pollutants; further inves-tigations such as effect-directed analyses are needed toidentify the unknown pollutants causing the high AhR-mediated activity. Furthermore, other investigations withinthe weight-of-evidence approach addressing the fish popula-tion decline in the upper Danube river also demonstrateda comparatively high hazard potential of sediments(M. Böttcher, S. Grund, S. Keiter, N. Seitz, H. Hollert andT. Braunbeck T, unpublished results; S. Grund, M. Böttcher,S. Keiter, K.Wurm, G. Reifferscheid,W.Manz, B. van Bavel,L. Erdinger, T. Braunbeck and H. Hollert, unpublished re-sults; N. Seitz, M. Böttcher, S. Keiter, T. Kosmehl, W. Manz,H. Hollert and T. Braunbeck, unpublished results).

Conclusion

In order to calculate the portion of unknown substancescausing high inductions in the DR-CALUX and the EROD

assay, these two biotest systems in combination withmultilayer fractionation and chemical analyses representsuitable tools to assess dioxin-like activity in sediments.The GPC.2D assay was only applied with crude extractsand revealed no correlation to the other bioassays. How-ever, to improve the comparability with other AhR-basedbioassays, there is a need to determine relative potencyfactors for the GPC.2D assay, testing several prioritypollutants.

In general, it is very difficult to prove that contaminationwith certain pollutants could have caused the decline of thefish population in the upper Danube river. Nevertheless,owing to the results of the bioanalytical tools applied,multilayer fractionations and chemical analysis a linkbetween chemical contamination and the decline of fishcatch in the upper Danube river cannot be ruled out. Inparticular, the high concentrations of PAHs with affinity tothe AhR in several samples might be one reason for fishpopulation decline. Several other studies showed that PAHcontamination is responsible for carcinogenicity, skin andliver tumor induction, immunosuppression, toxic effects oncardiac conduction and endocrine dysfunction in fish [15,26–29, 60]. However, the levels of the chemically deter-mined toxicants would not be a sole and sufficientexplanation for the decline of the fish population in theupper Danube river. On the other hand, in this investigationthe high AhR-mediated activities in a battery of bioanalyt-ical methods could not be completely explained bychemical analysis of priority pollutants. Thus, toxic impactof unknown nonpriority chemicals on natural fish popula-tions cannot be excluded. Therefore, further investigationwill have to focus on (1) bioassay-directed fractionationtechniques to identify pollutants responsible for the highdioxin-like activity as well as (2) ultrastructural alterationsin the liver of selected fish species and the correlation todioxin-like activities caused by river sediments.

Acknowledgements This study was supported by the GermanFederal Institute of Hydrology, Koblenz, and a German–Swedishexchange program funded by the German Academic Exchange Service(DAAD) and the Swedish Foundation for International Cooperation inResearch and Higher Education (STINT). Melanie Böttcher, NadjaSeitz and Karl Wurm contributed significantly to this article.

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Activities and identification of aryl hydrocarbon receptor agonists in sediments from the Danube...

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