A bioassay approach to determine the dioxin-like activity in sediment extracts from the Danube River: Ethoxyresorufin-O-deethylase induction in gill filaments and liver of three-spined sticklebacks (Gasterosteus aculeatus L.)

A bioassay approach to determine the dioxin-like activity in sediment extracts from the Danube River: Ethoxyresorufin-O-deethylase induction in gill filaments and liver of three-spined sticklebacks (Gasterosteus aculeatus L.)
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  A bioassay approach to determine the dioxin-like activity in sediment extracts fromthe Danube River: Ethoxyresoru 󿬁 n- O -deethylase induction in gill  󿬁 laments and liverof three-spined sticklebacks ( Gasterosteus aculeatus  L.)  Jens C. Otte a,b, ⁎ , Carin Andersson a , Alexandra Abrahamson a , Helena Olsman c , Steffen Keiter b ,Magnus Engwall c , Henner Hollert b,d , Björn Brunström a a Department of Environmental Toxicology, Evolutionary Biology Centre, Uppsala University, Norbyvägen 18A, 75236 Uppsala, Sweden b  Aquatic Ecology and Toxicology Section, Department of Zoology, University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany c Man-Technology-Environment Research Centre, Department of Natural Sciences, Örebro University, 70182 Örebro, Sweden d Department for Ecosystem Analyses, Institute for Environmental Research (Biology V), RWTH Aachen University, Worringerweg 1, 52074 Aachen, Germany A B S T R A C TA R T I C L E I N F O  Article history: Received 2 January 2008Accepted 14 May 2008Available online 20 June 2008 Keywords: EROD Gasterosteus aculeatus GillLiverSediment Sediment samples from the upper Danube River in Germany have previously been characterized asecotoxicologically hazardous and contaminants in these sediments maycontribute to the observed decline of  󿬁 sh populations in this river section. For the investigation of sediment toxicity there is a need fordevelopment, standardization and implementation of   in vivo  test systems using vertebrates. Therefore, themain objective of this study was to apply and evaluate a recently established  󿬁 sh gill EROD assay as abiomarker in sediment toxicity assessment by using extracts of well characterised sediment samples fromthe upper Danube River. This to our knowledge is the  󿬁 rst application of this novel assay to sedimentextracts. Sediments from four different sites along the upper Danube River were Soxhlet-extracted withacetone and dissolved in DMSO. Three-spined sticklebacks ( Gasterosteus aculeatus  L.) were exposed for 48 hto various concentrations of the extracts, to the positive control  β -naphtho 󿬂 avone or to the solvent.Measurements of EROD activity in gill  󿬁 laments and liver microsomes followed the exposure. Concentration-dependent induction of EROD in both gill and liver was found for all sediment extracts. The highest EROD-inducing potency was determined for extracts of sediments from the sites  “ Öp 󿬁 nger See ”  and  “ Sigmaringen ” and the EROD activities in gill and liver correlated well. The results from the gill and liver assays were inaccordance with  in vitro  results of previous investigations. The EROD activities measured in the present studycorresponded with the concentrations of PAHs, PCBs and PCDD/Fs in the sediment samples derived in aprevious study. The sticklebacks in this study were in the reproductive phase and a stronger EROD inductionwas obtained in the females than in the males. Implementation of the EROD assay in testing of sedimentextracts gave highly reliable results which make this assay an ecotoxicologically relevant method forassessment of contamination with Ah receptor agonists in sediments.© 2008 Elsevier Ltd. All rights reserved. 1. Introduction Lipophilic pollutants are ubiquitous in aqueous environments andare potentially hazardous to ecosystems and human health. Surfacewaters and sediments are often contaminated with complex mixturesof toxicants which can be present in high concentrations. Thereby,sediments may function as sinks for moderately and stronglylipophilic pollutants, but also as sources of such pollutants throughresuspension of particulate matter (Burton,1991; Brack, 2003; Viguriet al.,2007). Moderatelyandstronglylipohilicpollutantsof sedimentsinclude, e.g., polycyclic aromatic hydrocarbons (PAHs), polychlori-nated biphenyls (PCBs) and polychlorinated dibenzo-  p -dioxins(PCDDs) and dibenzofurans (PCDFs) (Engwall et al., 1999; Brack et al.,2005).Endo- and epibenthic organisms such as oligochaetae or bottom-dwelling  󿬁 sh like  󿬂 ounders and barbels are directly exposed tocontaminantsofsedimentarysrcin(Hellouetal.,1994;Goksøyretal.,1996; Egeler et al., 2001; Micheletti et al., 2007). Contact with, as wellas ingestion of, contaminants absorbed on sediment particles ordissolvedinporewatermayresultintheiraccumulationinorganisms.Toxic effects can occur in the exposed organism (Bouché et al., 2000) and trophic transfer may constitute a threat to pelagic organisms(Egeler et al., 2001). Another threat to those organisms may occur Environment International 34 (2008) 1176 – 1184 ⁎  Corresponding author. Aquatic Ecology and Toxicology Section, Department of Zoology, University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg,Germany. Tel.: +49 6221 546254; fax: +49 6221 546162. E-mail address: (J.C. Otte).0160-4120/$  –  see front matter © 2008 Elsevier Ltd. All rights reserved.doi:10.1016/j.envint.2008.05.004 Contents lists available at ScienceDirect Environment International  journal homepage:  when sediments containing contaminants are resuspended, whichleads to contamination of the water column (Westrich and Forstner,2005;Schwartzetal.,2006;Casado-Martínezetal.,2007;Gerbersdorf et al., 2007). Resuspension takes place from natural events such asbioturbation,  󿬂 ood or tide and from anthropogenic events, e.g.dredging, and comes along with higher bioavailability and accumula-tion of contaminants by pelagic organisms (Hollert et al., 2000;Geffard et al., 2002; Spencer et al., 2006). Main uptake sites forxenobiotics in 󿬁 sh are the gill and the gastrointestinal tract. Especiallythegill,asaninterfacebetweeninternalandexternalmilieu,isofhighrelevancy (Stein et al.,1984; McKim et al.,1985). The gill morphology does not only provide high ef  󿬁 ciency for gas exchange (Moyle andChech, 2000), but also for uptake of chemicals by passive diffusion.Certain chemicals, such as PAHs, PCBs and PCDD/Fs, inducecytochrome P450 1A (CYP1A) by ligand-activation of the aryl hydro-carbonreceptor(AhR).ThereisstrongevidencethatCYP1A-inductionin 󿬁 sh is dose-dependently related to levels of halogenated and non-halogenatedaromaticcompoundsintheorganismandtheenvironment(Guiney et al.,1997; Giesy et al., 2002), and several stages of the AhR- mediated pathway have been used or proposed as biomarkers in theassessmentof thesecompounds(GoksøyrandFörlin,1992;BucheliandFent, 1995; Behnisch et al., 2001; van der Oost et al., 2003). Hence,induction of CYP1A is one of the best studied biomarkers for environ-mental contamination in aquatic ecosystems and it is often quanti 󿬁 edusing enzyme assays, especially measurement of 7-ethoxyresoru 󿬁 n- O -deethylase (EROD) activity (Whyte et al., 2000; Hollert et al., 2002;Kammann et al., 2005). Compared to other endpoints of the AhR-mediated pathway, the EROD activity is a comparatively sensitive tooland it mayevenbeanindicator for effects atvariouslevels of biologicalorganization(vanderOostetal.,2003).Asabiomarker,ERODactivityin 󿬁 shisprimarilyassessedintheliver,eventhoughinductionofCYP1Aisnot only localized to hepatic tissue (Husøy et al., 1994; Carlsson et al.,1999; Whyte et al., 2000). Biomarkers in tissues which are in contactwith the external environment, for example the gill, are of stronginterestinthecontextofbiomonitoringandriskassessment(LevineandOris1999).AmethodtomeasureERODactivityin 󿬁 shgill 󿬁 lamentswasrecently described ( Jönsson et al., 2002) and has been applied and evaluatedasabiomarkertoassesswaterbornepollutants( Jönssonetal.,2003, 2004; Andersson et al., 2006; Mdegela et al., 2006). Additionally,Ahlf et al. (2002) and Hollert et al. (2003) stated the urgent need for the development, standardization and implementation of   in vivo  testsystems or biomarkers in vertebrates for the investigation of sedimenttoxicity. Therefore, the main objective of this study was to apply thegill EROD assay as a vertebrate-based biomarker in sediment toxicityassessment.Sediment samples for this investigation were from the upperDanube River, Germany. These samples had previously been char-acterizedasecotoxicologicallyhazardousandaspotentiallycontribut-ing to a decline of   󿬁 sh populations in the upper Danube River (Keiteretal.,2006).Afollowingintegratedapproachusingtriadinvestigations(Chapman, 2000; Chapman and Hollert, 2006) was carried out andfocused on chemical analysis of water quality and on monitoring of  Fig. 1.  Overview of the sampling sites along the Upper Danube in Germany. a) Sigmaringen, b) Riedlingen, c) Öp 󿬁 nger See, d) Bad Abbach (Keiter et al., 2008, modi 󿬁 ed). Fig. 2. MeanERODactivityingill(a)andliver(b)insticklebacks( n =6)exposedtoDMSO(100ppm),toaprocesscontrol(100 μ  lDMSOsolution/litretestmedium)andto β -NF(1 μ  M).A probability value of   p ≤ 0.01 is indicated by two asterisks. PC=process control.1177  J.C. Otte et al. / Environment International 34 (2008) 1176  – 1184  different hydrological and biological parameters (Boettcher et al.,submitted for publication; Keiter et al., 2008; Seitz et al., 2008). High dioxin-likeactivitywasassessed invitro forsedimentsatdifferentsitesalong the river course using the cell lines GPC.2D.Luc, H4IIE (DR-CALUX®)andRTL-W1.Comparisonof theconcentrationsofpersistent(PCDDs, PCDFs and PCBs) and non-persistent (PAHs) organic com-poundswiththetotal dioxin-likeactivitymeasured  invitro  ledtoonlypartial explanation of the obtained activity (Keiter et al., 2008). Fig. 3.  EROD activity in gill (a) and liver (b) after exposure to sediment extracts from the sites  “ Sigmaringen ” ,  “ Riedlingen ” ,  “ Öp 󿬁 nger See ”  and  “ Bad Abbach ” . Sticklebacks ( n =5 – 6)wereexposedfor48hunderstaticconditionstosedimentextractsatthesedimentequivalentconcentrationsof4mg/l,0.8mg/l,0.16mg/lor0.032mg/lortoapositivecontrol(1 μ  M β -NF) or a solvent control (100 ppm DMSO). Activities that differ signi 󿬁 cantly from the solvent control are indicated by one (  p ≤ 0.05) or by two (  p ≤ 0.01) asterisks.1178  J.C. Otte et al. / Environment International 34 (2008) 1176  – 1184  In this study, EROD induction in sticklebacks  in vivo  was evaluatedas a biomarker in sediment toxicity assessment by comparing ourresults in gill and liver with previous data on the  in vitro  ERODinduction by extracts of the same sediment samples and withconcentrations of PAHs, PCBs and PCDD/Fs in those extracts.Furthermore, the relevancy of the measured EROD activities for thedeclining  󿬁 sh populations in the upper Danube River is discussed.Thethree-spinedstickleback( Gasterosteusaculeatus L.)wasselectedfor this study because of its high ecological relevance in temperate andboreal climate zones of the northern hemisphere (Paepke, 2001) andbecauseofitssmallsizeimplicatingsmalltestingvolumes.Itssuitabilityasamodelorganisminecotoxicologyhasalreadybeendemonstratedinanumberofstudies(e.g.Holmetal.,1993;Bell,2001;Egeleretal.,2001;Katsiadakiet al., 2002; Hahlbeck et al.,2004a,b;Andersson et al., 2006)and it serves as a test species in standardised test guidelines in aquatictoxicology (Environment Canada,1990; OECD,1992). 2. Materials and methods  2.1. Sediment samples Nearsurfacesamples (0 – 5cmsedimentdepth)werecollectedalong theupper partof the Danube River at Sigmaringen, Riedlingen, Öp 󿬁 nger See and Bad Abbach (Fig.1).Depending on local conditions, the sediment samples at  “ Öp 󿬁 nger See ”  and  “ BadAbbach ”  were taken with a Van-Veen gripper and at  “ Sigmaringen ”  and  “ Riedlingen ” with a stainless steel shovel. To compensate for variation in concentration of contaminants within the sampling site, several samples were taken at each site andpooled and homogenised. The surface sediments at Sigmaringen and Riedlingen wereboth sampled in May, the sediments at Öp 󿬁 nger See in July and the sediments at BadAbbach during October the same year. The riversection at Sigmaringen is not regulatedand meanders whereas the section at Riedlingen is characterized by canalization and aresulting high streamvelocity. The sampling site  “ Öp 󿬁 nger See “  forms part of a storagereservoir along the river course which shows a high sedimentation rate and tends tobecome a shallow water. The river section at the sampling site  “ Bad Abbach ”  is part of the navigable waterways.The samples were cooled and transported to the laboratory, immediately frozen to − 20 °C, freeze dried by using a freeze-drying machine (Alpha 1 – 4, Christ, Osterode,Germany) and stored in glass bottles (Duran, Schott, Mainz, Germany) at 4 °C untilextraction the year after. Dried sediment was transferred to extraction thimbles(200ml, Schleicher &Schuell, Dassel, Germany)in 20-g portionsand thethimbleswereplaced in Soxhlet extractors (Schott, Mainz, Germany). Extraction with approximately350 ml acetone p.a. (Riedel-de Haën, Seelze, Germany) was allowed to proceed at 7 – 9cycles per hour for 16 h under permanent water cooling. After extraction, each extractwasreducedtoavolume ofapproximately5 ml ina rotaryevaporator(Rotavapor R110;Büchi Laborations-Technik AG, Flawil, Switzerland) at 38 °C and at low pressure byusing a water-jet vacuum pump. Following transfer to a vial, 0.5 ml of dimethylsulfoxide (DMSO; Sigma-Aldrich, Steinheim, Germany) was added. The acetone wasevaporated at approximately 38 °C using a nitrogen stream and the DMSO solutionwassonicated. The compounds extracted from 20 g dry sediment were by this proceduredissolved in 0.5 ml DMSO (40 g sediment equivalents per ml DMSO). To study thepossibility of contamination by extraction, an empty thimble was treated exactly thesame way as described above (process control). Any compounds extracted weredissolvedin0.5mlDMSOandtestedalongwiththesedimentextracts.Allextractswerestored at 4 °C in darkness.  2.2. The species under study Adultthree-spined sticklebacks were caught in March 2004on the Swedishsouth – west coast. The  󿬁 sh used in the experiments had been held in tanks in the aquariumfacility at the Evolutionary Biology Centre, Uppsala University (Uppsala, Sweden) for atleast2years.ThetankswerecontinuouslysuppliedwithaeratedUppsalatapwaterandthe photoperiod was set to 8 h light and 16 h darkness. Some weeks before theexperiments in March, the  󿬁 sh were relocated to a glass aquarium and the daylightcycle was adjusted automatically to the diurnal variations at latitude 52 °N. The  󿬁 shwerefed  Artemiaspec. onceaday.The 󿬁 shwerenotfedfrom2daysbeforetheinitiationof exposure and throughout exposure.  2.3. Experimental design The individual exposures of the sticklebacks were processed during a 2-weekperiod 18 days after the relocation to the glass aquarium. Copper-free tap water wasbottled and aerated for 2 days at room temperature before the exposure was started.The exposure was done using 2 l borosilicate glass beakers (VWR International,Stockholm, Sweden)  󿬁 lled with 1 l of the prepared tap water. The sediment extractswereaddedtothewaterinconcentrationsof4mgsedimentequivalents(SEQ)/l,0.8mgSEQ/l and 0.16 mg SEQ/l. Two samples were also tested at 0.032 mg SEQ/l.  β -Naphtho 󿬂 avone ( β -NF) was also dissolved in DMSO and added to the water to yield1  μ  M of   β -NF (50 ppm DMSO). All samples were tested in two replicates along with thepositive control ( β -NF) and a solvent control (the highest solvent concentration used,100 ppm DMSO). Each replicate consisted of 3  󿬁 sh which were placed in the beakersand exposed for 48 h. The water was aerated continuously byaquarium pumps and thebeakers were covered and kept in darkness to avoid photolytic degradation. After 48 h,the  󿬁 sh were anesthetized in a solution of ethyl-  p -aminobenzoate (Sigma, Germany),killed by cranial dislocation and gill arches and liver were carefully removed. Liversamples were frozen to  − 80 °C and the gill arches were transferred to ice-cold HEPES-Cortland buffer (pH 7.7) for EROD analysis. Furthermore, the nitrite and ammoniaconcentrations in the water were controlled and found to be below 0.1 and 0.02 mg/l,respectively. These concentrations should not affect the  󿬁 sh (Andrews et al.,1988).  2.4. Measurement of EROD activity in the gill The procedure described by Jönsson et al. (2002) for rainbow trout, adapted tosticklebacksbyAnderssonetal.(2007),wasusedfordeterminationoftheERODactivityin gill  󿬁 laments. The gill  󿬁 laments were cut off from the cartilage part of the gill archesand triplicates of about 15  󿬁 laments were arranged in a 12-well tissue culture plate.Followingstepsweredoneunderlight-protectedconditions.Reactionbuffer,consistingof1 μ  M7-ethoxyresoru 󿬁 nand20 μ  MdicumarolinHEPES-Cortlandbuffer,wasaddedineach well. The incubation took place under constant shaking for 10 min at roomtemperature.Subsequently,thereactionbufferwasrenewedandincubationproceeded.After 40 and 60 min, 0.2-ml samples were transferred from each well to a Fluoronunc Fig. 5.  EROD activity in gill (a) and liver (b) in males and females after exposure to thesolvent control (100 ppm DMSO) as well as in gill (c) and liver (d) of males and femalesafterexposuretothepositivecontrol(1 μ  M β -NF).Thenumberofsticklebackswaseightfor each sex for the exposure to the solvent and 14 – 18 for each sex for the positivecontrol. Signi 󿬁 cant differences between the sexes are indicated by one (  p ≤ 0.05) or bythree (  p ≤ 0.001) asterisks. Fig. 4.  Lowest observed effect concentrations (LOECs) for induction of gill and liverEROD activities for the four sites investigated. SEQ=Sediment equivalents.1179  J.C. Otte et al. / Environment International 34 (2008) 1176  – 1184  96-well plate (Nunc A/S, Roskilde, Denmark). The deethylation of exogenous 7-ethoxyresoru 󿬁 n to form resoru 󿬁 n was measured using the wavelengths 544 nm (ex)and 590 nm (em). Aliquots of resoru 󿬁 n standard solutions (0 – 250 nM) were preparedwith reaction buffer from a 0.1 mM stock solution in methanol. EROD activity wascalculated and expressed as picomole resoru 󿬁 n per  󿬁 lament and minute.  2.5. Measurement of EROD activity in the liver  Within 4 weeks after sampling of the tissue, EROD activity in the liver was measuredaccordingtoamethoddescribedbyKennedyandJones(1994).Theliverwashomogenized in ice-cold homogenization buffer (0.15 M KCl, 1 mM EDTA in 0.1 M phosphate buffer;pH7.4) using a Potter-Elvehjem homogeniser(B. Braun, Melsungen, Germany)with 1200revolutions per minute. The homogenate was centrifuged at 10000  g   (Rotanda 460R;HettichZentrifugen,Tuttlingen,Germany)for15min(4°C)andthenthesupernatantwastransferredandcentrifugedagain(L8-70ultracentrifuge;BeckmanInstruments,Fullerton,CA, USA) at 105000  g   for 1 h (4 °C). The resulting pellet, containing the microsomes, wasresuspendedin1mlHEPES-Cortlandbuffer(pH8.0)and,ifnecessary,thesuspensionwasdiluted in the same buffer. Standard solutions of resoru 󿬁 n (0 – 500 nM) and bovine serumalbumin (0 – 6 mg/ml) were also made in HEPES-Cortland buffer. For each microsomesampleandeachconcentration ofresoru 󿬁 nstandard,45- μ  lsampleswere transferredto aFluoronunc96-wellplate(NuncA/S,Roskilde,Denmark)astriplicates.Subsequently,160 μ  lof a reaction solution (15  μ  M 7-ethoxyresoru 󿬁 n and 2.1 mM NADPH in HEPES-Cortlandbuffer) was added to all wells and the  󿬂 uorescence was immediately measured in aWallac3platereader(WallacOy,Turku,Finland)(excitation544nm,emission590nm)andseveraltimeswithin20min.Todeterminetheproteinconcentration,45- μ  laliquotsofeachmicrosome suspension and of the protein standard were added to a Fluoronunc 96-well Fig. 6.  EROD activity in gill (a) and liver (b) after exposure to sediment extracts from the sites  “ Sigmaringen ” ,  “ Riedlingen ” ,  “ Öp 󿬁 nger See ”  and  “ Bad Abbach ” . Data is shown as split(male/female) and mixed groups.1180  J.C. Otte et al. / Environment International 34 (2008) 1176  – 1184
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