Delayed effects of environmentally relevant concentrations of 3,3′,4,4′-tetrachlorobiphenyl (PCB77) and non-polar sediment extracts detected in the prolonged-FETAX

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Delayed effects of environmentally relevant concentrations of 3,3′,4,4′-tetrachlorobiphenyl (PCB77) and non-polar sediment extracts detected in the prolonged-FETAX

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  Delayed effects of environmentally relevant concentrations of 3,3 ′ ,4,4 ′ -tetrachlorobiphenyl (PCB-77) and non-polar sediment extracts detected in the prolonged-FETAX Arno C. Gutleb  1 , Leonie Mossink, Merijn Schriks, H.J.H. van den Berg,Albertinka J. Murk  ⁎ Toxicology Section, Wageningen University, Tuinlaan 5 NL-6703 HE Wageningen, The Netherlands Received 1 December 2006; received in revised form 2 February 2007; accepted 1 March 2007Available online 23 April 2007 Abstract In the prolonged-FETAX (prolonged-Frog Embryo Teratogenic Assay-  Xenopus ) tadpoles are allowed to develop untilmetamorphosis after an initial 4 day early life-stage exposure (FETAX). PCB 77 (3,4,3 ′ ,4 ′ -tetrachlorobiphenyl) and sediment extracts were used in the presented experiments. Concentrations of PCB 77 (0.03 nM=8.55 ng/L; 3 nM=855 ng/L;300 nM=85.5  μ g/L, 30,000 nM=8.55 mg/L) were derived on the basis of dioxin-like toxic equivalency concentrations known to be present in pore-water. The results were compared to those obtained with the standard FETAX.Intheprolonged-FETAXthepercentageofanimalspassingmetamorphosiswithin115dayswassignificantlydecreasedinthegroupsexposed to 300 or 30,000 nM PCB 77. Significantly increased percentages of tadpoles were halted in thyroid hormone dependent earlymetamorphicNFstages ≤ 55 (3nM PCB 77) and metamorphic stages 56 – 60tadpoles (300nM PCB 77and30,000nM PCB 77).Non- polar sediment extracts from two contaminated Dutch sites significantly decreased, and one significantly increased the percentage of animals that passed metamorphosis. These results were in accordance with earlier in vitro results in a thyroid hormone dependent assayandstronglysuggestthepresenceofthyroidhormonedisruptingcompoundsintheaquaticenvironment.IntheclassicalFETAXneither PCB 77 nor non-polar sediment extracts induced any effects. The results of the present study indicate that the prolonged-FETAX is animportant in vivo tool in addition to the in vitro T-screen to assess long-term effects of early life-stage exposure to environmentallyrelevant concentrations of (mixtures of) compounds and that affect thyroid hormone dependent physiology.© 2007 Elsevier B.V. All rights reserved.  Keywords:  Prolonged-FETAX; Amphibians; Development; Bioassay 1. Introduction The Frog Embryo Teratogenesis Assay-  Xenopus (FETAX) test, a short-term early-life-stage test has beenshown to be a powerful bioassay for the identification of compounds that exhibit embryotoxic potency (Dumont et al., 1983; Fort and Bantle, 1990; Luo et al., 1993;Bantle et al., 1994a,b; Bernardini et al., 1996; Herkovitset al., 1997; Fort et al., 2000; Mann and Bidwell, 2000; Science of the Total Environment 381 (2007) 307 – 315www.elsevier.com/locate/scitotenv ⁎  Corresponding author. Tel.: +31 317 48 32 33.  E-mail addresses:  arno.gutleb@veths.no (A.C. Gutleb),Tinka.Murk@WUR.nl (A.J. Murk). 1 Current address: Department of Production Animal ClinicalScience Norwegian School of Veterinary Science POB 8146 Dep. N-0033 Oslo Norway.0048-9697/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.scitotenv.2007.03.002  Fort and Paul, 2002). Nevertheless the FETAX has beenshown to underestimate long-term effects of persistent compounds with low acute embryotoxicity such as polychlorinated biphenyls (PCBs) that were detected bythe prolonged-FETAX (Gutleb et al., 1999). No effect onmortality, rate of malformations, growth, or development of   Xenopus laevis  embryos was observed with theFETAX scored on day 4 at Nieuwkoop and Faber NFstage 46 ( Nieuwkoop and Faber, 1975) in a concentrationrange up to 15.3  μ M (5 mg/L) PCB 126 (Gutleb et al.,1999). However, when the animals were not sacrificedafter4daysofexposurebutratherplacedoverintoaquariaand kept unexposed until they metamorphosed, a dose-dependent increase in mortality and number of animalsshowing malformations was observed at exposure con-centrationsaslowas17.2pM(5.6ng/L)PCB-126(Gutlebet al., 1999) or 1.7 fmol TCDD-equivalents (TEQ)/mLusing a relative potency (TEF) of 0.1. These concentra-tions are much lower than the levels up to 317 fmol TEQ/ mLpore-waterobservedinthemostpollutedsedimentsinthe Netherlands tested in the CALUX assay (Murk et al.,1996). Pore-water reflects the environment in which anamphibian embryo is exposed in close contact withsediment (Karasov et al., 2005).This study wanted to test the effect of a readily metab-olizable congener PCB 77 (3,4,3 ′ ,4 ′ -tetrachlorobiphenyl)(Murk et al., 1994) and compare the effects on a TEQ basisusingarelativepotency(TEF)of0.001withthoseof the sparingly metabolizable PCB 126 mentioned above.In addition real sediment extracts from some Dutchsites polluted with a wide range of PHAHs and PAHs(Vethaak et al., 2002) were tested for their potency toinduce serious effects in the prolonged-FETAX. Theextracts had been tested before in the T-screen andaltered thyroid hormone dependent cell growth in anvitro proliferation screen (Gutleb et al., 2005). 2. Animals, materials and methods 2.1. Chemicals PCB 77 (99% purity) was obtained from Promochem(Wesel, Germany). Human chorionic gonadotropichormone (hCG) was obtained from Organon (Oss, The Netherlands). All other chemicals used throughout theexperiments were of analytical grade and obtained fromMerck (Darmstadt, Germany). 2.2. Preparation of sediment extracts Twosedimentsampleswerefromknownpollutedsitesin the Netherlands, namely the Westerschelde close toTerneuzen,andthe Dommel close toEindhoven(Vethaak et al., 2002). In addition sediment samples were collectedfrom two locations on a small channel close toHoogeveen, where malformed  Rana temporaria  tadpoleswere observed in August 2000. Tadpole metamorphosisin this channel was delayed by at least 2 months andtadpoleshadveryshortbutthickhindlegsthatwerepartlyrotated in the joints (unpubl. results). Non-polar fractionsof sediment were obtained as previously described(Legler et al., 2002). Briefly portions of sediment (10×2 g) were vortexed (1 min) after the addition of 2 mL of acetone and allowed to stand for 10 min prior tovortexing (1 min) again. A mixture (4 mL) of hexane/ diethylether(Hx-DEE,9:1)wasaddedandaftervortexing(1 min) samples were centrifuged for 2 min (400 ×g) andthe upper layer of all ten parallel extracts was transferredinto pre-washed glass tubes. This step was performed 3times after which 200  μ L Hx-DEE was added and theupper layer was collected again. Solvent was evaporatedat 30 °C under a gentle stream of nitrogen. Prior tocomplete evaporation the ten samples were quantitatively pooled in a single vial, slightly warmed and kept under agentle stream of nitrogen until dryness. Hexane (500  μ L)was added and the sample was filtered over a 1 g Na 2 SO 4 column, evaporated and resuspended in 20  μ L DMSOresulting in a non-polar fraction. 2.3. Animals, breeding and housing  All experiments were performed at the ToxicologySection, Wageningen University and Research Centre.AdultAfricanclawedfrogs(  X.laevis )wereobtainedfromthe Department for Experimental Zoology, CatholicUniversity of Nijmegen. All experimental animals werekept at a 12:12 photoperiod schedule. Adult   X. laevis were maintained in aquaria with constantly filtered andaerated water at 20 °C. Details of breeding conditionshavebeenreportedpreviously(Gutlebetal.,1999,2000).Developmentalstageofembryosandtadpoleswasscoredaccording to Nieuwkoop and Faber (1975). 2.4. Experimental protocol  FETAX exposure followed the srcinal protocol(ASTM, 1991) with few modifications. Rather than performing duplicates with embryos from 3 different adult pairs fertilized embryos from 3 pairs were pooled before embryo selection. FETAX medium was usedthroughout FETAX exposure after which tap water wasused in the prolonged-FETAX. ASTM (1991) controlmortalityandmalformationcriteriaoftestacceptancewereapplied but no positive control compounds were used. 308  A.C. Gutleb et al. / Science of the Total Environment 381 (2007) 307   –  315  DMSO was used as a solvent in the FETAX exposures at 0.5% in the water phase. The exposure medium wasrenewed every day. The FETAX and prolonged-FETAXexposures always were performed in quadruplicate (4×25embryos starting at NF stage 8 – 11) at 24 °C and lasted for 96-h when embryos had reached NF stage 46. 2.4.1. Experiment 1 Embryos were exposed to PCB 77 (nominal concen-trations: 0.03 nM=8.55 ng/L; 3 nM=855 ng/L;300 nM=85.5  μ g/L, 30,000 nM=8.55 mg/L) or toDMSO in quadruplicate. The TEQ-concentrations of these exposures are 30 fmol/L – 30 nmol/L. 2.4.2. Experiment 2 Embryos were exposed in quadruplicate to non-polar sedimentextracts(equivalentto250mgsedimentin10mLexposure medium) or to DMSO as a negative control.Embryos from two randomly selected groups of thefourexposuregroupswereassignedfortheFETAX.Theywere anaesthetised in ice-water and thereafter fixed informalin (4%) for stereomicroscopic examination after 96 h when animals have reached NF stage 46. For the prolonged-FETAX the tadpoles of the remaining dupli-cates (NF stage 46) were placed in aquaria of 20×20×30 cm (400 mL water/animal). The aquariawere checked daily for the presence of dead animals andtadpoles found dead were fixed and scored for malforma-tions (Bantle et al., 1991). Tadpoles were fed a diet consisting of 500 g dried nettle powder (Jacob Hooy,Limmen,TheNetherlands),5gcoffee-creamer(FrieslandDairy Foods, Leeuwarden, The Netherlands), 5 g agar granulated and 5 g yeast extract (both from BectonDickinson, Cockeysville, MD, USA). The powder (200 mg) was mixed with tap water to yield a viscousmixture, which was added once a day to the aquaria.Water from all exposure experiments was charcoal-filteredpriortodisposal.Roomtemperatureinexperiment 1 was 21 °C and 24 °C in experiment 2 to acceleratedevelopmental speed.Animals reaching metamorphic NF stage 65/66 wereanaesthetised in ice-water and thereafter sacrificed bycervical trans-section, weighed, scored for malformations(oedema, eye and leg deformations, depigmentation) andwrappedinaluminiumfoilandstoredat  − 20°C.NFstage65 was chosen as the final stage for our goal as thedisappearanceofthetailremnantsthatmarkthedifference between NF stage 65 and NF stage 66 can vary over several days and is not dependent on changes of thyroidhormone. As tadpole growth and development is knownto be density dependent (Werner, 1986; Scott, 1994),water volume was kept constant per animal by removing400mLperanimaltakenoutofanaquarium.Attheendof the 115-day experimental period, the remaining tadpoleswere anaesthetised in ice-water, sacrificed by cervicaltrans-section and their developmental stage was scored( Nieuwkoop and Faber, 1975).To be able to interpret the data in different ways theresults are presented in three different ways startingsimply by comparing percentages of animals that passedmetamorphosis within the experimental period as defined by reaching NF stage 65. Secondly tadpoles are rankedinto three categories based on the thyroid hormonedependency of their developmental stage. The threecategories were: stages before thyroid hormone depen-dency (NF stages  ≤ 55; early metamorphic or premeta-morphic), peak thyroid hormone dependent stages (NFstages 56 – 60, metamorphic or prometamorphic) and latemetamorphic stages (NF stages 61 – 65; postmetamorphicor climax stages) that show decreasing thyroid hormonelevels (Etkin, 1932). Thirdly, as the number of animalsthatdidnotreachthemetamorphosisdoesnotindicatetheseverity of their retardation, also penalty points areaccredited to each animal per group based on the number of developmental stages possibly lacking until finalisingmetamorphosis.Animalswereaccredited0penaltypointswhentheyhadreachedNFstage65,1forNFstage64andsoforth.Theuseofpenaltypointsisdescribedforthefirst time in this manuscript. 2.5. Data analysis All data are reported as means±standard error of themean(SEM).Differencesbetweenmeansweretestedwithone-way ANOVA or Kruskal – Wallis  H  -Test whereappropriate after testing the data for normality. Theacceptance level was set at   P  b 0.05. Statistical analyseswere performed using SPSS/PC+, version 6.0 (SPSS Inc., Table 1 Number of animals ( n ) of the total number of surviving animals (  N  )that had reached NF stage 65 after 115 days (water temperature 21 °C),their average body weight (mg±SEM) and length of larval period(days±SEM) after early exposure during the first 96 h (NF stage 8-46)to PCB 77 according to the prolonged-FETAX-protocolPCB 77 (nM)  n /   N   Body weight (mg±SEM)Larval period(days±SEM)0 31/48 180±9 93.2±30.03 31/46 167±7 87.2±33 a  15/22 205±6 96.2±2300 19/46 ⁎  215±14 93.3±330,000 17/45 ⁎ 168±8 93.3±3 a  One group of the duplicates was terminated earlier due a decrease inwater quality (data not included). ⁎  P  N 0.05.309  A.C. Gutleb et al. / Science of the Total Environment 381 (2007) 307   –  315  Chicago, IL, USA). Statistical evaluation for the percen-tagesofanimalsthatpassedmetamorphosisandthestagesof tadpoles that did not pass metamorphosis werecomparedbetweentreatmentsby χ 2contingencyanalysisusing GraphPad Prism 4 (San Diego, CA, USA). Statis-tical evaluation (body weight, larval period) was made byone-way ANOVA, and pairwise comparisons of meanswithin significant treatments were made using Bonferro-ni's testthat controlledTypeI errors.Statistical evaluationof total penalty points/group and mean penalty points/ animal was made by Kruskal – Wallis  H  -Test,  ⁎  P  b 0.05. 3. Results 3.1. Experiment 13.1.1. FETAX  After 96-h exposure to any of the PCB 77 concen-trations 0.03 nM, 3 nM, 300 nM, 30,000 nM) lowmortality and rate of malformations were observed( b 10%) that were not dose-dependent and no significant effects on growth were observed with mean bodylengths for all groups ranging from 7.9 – 8.2 mm (datanot shown) (one-way ANOVA,  P  N 0.05). 3.1.2. Prolonged-FETAX  Animals in one group of the duplicates that had beenexposed to 3 nM PCB 77 were terminated during theexperimentalperiodafterhavingsufferedfroma decreasein water quality as a fungus affected the detritus in theaquarium. Results from this group are not included in thetables and figures. Mortality in any of the other exposuregroupswaslessthan15%andnomalformations(oedema,eye and leg deformations, depigmentation) occurred inany of the groups until day 115. Body weight at NF stage Fig. 1 (a) Percentage of tadpoles reaching metamorphosis after exposure to PCB 77 according to the prolonged-FETAX protocol. ( ⁎ )  P  b 0.1; ( ⁎ )  P  b 0.05. (b) Stage of tadpoles at the end of the 115 daysexperimental period after exposure to PCB 77 (nM) according prolonged-FETAX protocol. The percentage of tadpoles that did tothe finish metamorphosis in 115 days is indicated in this figure. ⁎  P  b 0.05. (c) Total penalty points/group and mean penalty points/ animal assigned to animals that did not reach the end of metamorphosis (NF stage 65) after 115 days of exposure at 21 Cafter exposure to PCB 77 according to the prolonged-FETAX protocol.Animals reaching NF stage 65 are attributed 0 penalty points, NF stage64 animals 1 and so forth. Statistical evaluation was made by Kruskal – Wallis  H  -Test,  ⁎  P  b 0.05.Table 2 Number of animals ( n ) of the total number of surviving animals (  N  )that had reached NF stage 65 after 115 days (water temperature 24 °C),their average body weight (mg±SEM) and length of their larval period(days±SEM) after early exposure during the first 96 h (NF stage 8-46)according to the p-FETAX protocol to non-polar sediment extracts(equivalent to 250 mg sediment in 10 mL exposure medium)Sediment extract   n /   N   Body weight (mg±SEM)Larval period(days±SEM)Control 29/46 234±14 76.6±6Dommel 23/47 ⁎ 223±14 81.2±3Terneuzen 20/48 ⁎  175±11 ⁎⁎  87.8±2 ⁎ Hoogeveen I a  18/23 ⁎  184±15 ( ⁎ ) 83.8±3Hoogeveen II 38/48 ⁎ 190±8 ( ⁎ ) 80.1±3 ( ⁎ )  P  b 0.1;  ⁎  P  b 0.05;  ⁎⁎  P  b 0.01. a  One group of the duplicates was terminated earlier due a decrease inwater quality (data not included).310  A.C. Gutleb et al. / Science of the Total Environment 381 (2007) 307   –  315  65/66 and duration of larval period until reaching NFstage 65 were not significantly different between any of the groups (Table 1).Early life-stage exposure to 300 nM and 30,000 nMPCB 77 resulted in significantly decreased percentage of animals that passed metamorphosis as compared withcontrol groups after 115 days ( χ 2 =36.85;  df   =4;  P  b 0.05), whereas after 60 days ( χ 2 =12.65;  df   =4;  P  N 0.05), 90 days ( χ 2 =12.80;  df   =4;  P  N 0.05), and100 days ( χ 2 =19.48;  df   =4;  P  N 0.05) no significant differences were found (Fig. 1a).In the groups exposed to 3 nM PCB 77 the percentageof tadpoles that were still early metamorphic (NFstages ≤ 55) on day 115 was significantly increasedcompared with control groups ( χ 2 =17.83,  df   =4;  P  =0.05). Exposure to 300 nM and 30,000 nM PCB 77resulted in significantly increased percentages of meta-morphic tadpoles ( χ 2 =21.18,  df   =4;  P  b 0.05) (NF stages56 – 60), whereas no such differences compared withcontrol groups were found for late metamorphic tadpolesfor any exposure concentration (NF stages 61 – 64) after 115 days in exposed groups ( χ 2 =19.20,  df   =4;  P  N 0.05)(Fig. 1 b).The number of penalty points awarded to prolonged-FETAX exposed tadpoles at the end of the experimental period was significantly increased for the groups exposedto 300 nM PCB 77 or higher compared with controlgroups (Kruskal – Wallis  H  -Test,  P  N 0.05) (Fig. 1c). 3.2. Experiment 23.2.1. FETAX  After 96-h exposure to non-polar sediment extracts(exposure equivalent to extract of 250 mg sediment/ 10mL)lowmortality( b 10%)andnomalformationswereobserved either in control groups and in non-polar sediment extracts exposed groups. Growth in the 96-hourexposureperiodwasnotaffectedinanygroup(mean body length ranging from 7.9 – 8.2 mm) (one-wayANOVA,  P  N 0.05). 3.2.2. Prolonged-FETAX  Animals in one group of the duplicates that had beenexposedtonon-polarsedimentextractsfromHoogeveenIwere terminated during the experimental period after having suffered from a decrease in water quality as afungus affected the detritus in the aquarium. Results fromthis group are not included in the tables and figures. Fig. 2 (a) Percentage of tadpoles reaching metamorphosis after exposure to non-polar sediment extracts. Statistical evaluation for the percentages of animals that passed metamorphosis was made by  χ 2 contingency analysis,  ⁎  P  b 0.05. (b) Stage of tadpoles at the end of the115 days experimental period after exposure to non-polar sediment extracts (equivalent to 250 mg sediment in 10 mL exposure medium).The animals were exposed according to the prolonged-FETAX protocol during 96 h from NF stage 8 to NF stage 46, and followedin their further development without any additional exposure. The percentage of tadpoles that did finish metamorphosis in 115 days wasindicated in Fig. 2a. Statistical evaluation for the distribution of animals into the three categories was made by χ 2 contingency analysis, ⁎  P  b 0.05. (c) Sediment-specific assigned penalty points. Total penalty points/group and mean penalty points/animal at the end of the 115 dayexperimental period. Animals reaching NF stage 65 were counted as 0, NF stage 64 was 1 and so forth. Statistical evaluation was made byKruskal – Wallis  H  -Test,  ⁎  P  b 0.05.311  A.C. Gutleb et al. / Science of the Total Environment 381 (2007) 307   –  315
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