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Genotoxic and teratogenic effect of freshwater sediment samples from the Rhine and Elbe River (Germany) in zebrafish embryo using a multi-endpoint testing strategy

  • Danio rerio as a Model in Aquatic Toxicology and Sediment Research
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Abstract

The embryotoxic potential of three model sediment samples with a distinct and well-characterized pollutant burden from the main German river basins Rhine and Elbe was investigated. The Fish Embryo Contact Test (FECT) in zebrafish (Danio rerio) was applied and submitted to further development to allow for a comprehensive risk assessment of such complex environmental samples. As particulate pollutants are constructive constituents of sediments, they underlay episodic source-sink dynamics, becoming available to benthic organisms. As bioavailability of xenobiotics is a crucial factor for ecotoxicological hazard, we focused on the direct particle-exposure pathway, evaluating throughput-capable endpoints and considering toxicokinetics. Fish embryo and larvae were exposed toward reconstituted (freeze-dried) sediment samples on a microcosm-scale experimental approach. A range of different developmental embryonic stages were considered to gain knowledge of potential correlations with metabolic competence during the early embryogenesis. Morphological, physiological, and molecular endpoints were investigated to elucidate induced adverse effects, placing particular emphasis on genomic instability, assessed by the in vivo comet assay. Flow cytometry was used to investigate the extent of induced cell death, since cytotoxicity can lead to confounding effects. The implementation of relative toxicity indices further provides inter-comparability between samples and related studies. All of the investigated sediments represent a significant ecotoxicological hazard by disrupting embryogenesis in zebrafish. Beside the induction of acute toxicity, morphological and physiological embryotoxic effects could be identified in a concentration-response manner. Increased DNA strand break frequency was detected after sediment contact in characteristic non-monotonic dose–response behavior due to overlapping cytotoxic effects. The embryonic zebrafish toxicity model along with the in vivo comet assay and molecular biomarker analysis should prospectively be considered to assess the ecotoxicological potential of sediments allowing for a comprehensive hazard ranking. In order to elucidate mode of action, novel techniques such as flow cytometry have been adopted and proved to be valuable tools for advanced risk assessment and management.

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References

  • Billiard SM, Meyer JN, Wassenberg DM et al (2008) Nonadditive effects of PAHs on early vertebrate development: mechanisms and implications for risk assessment. Toxicol Sci Off J Soc Toxicol 105:5–23. doi:10.1093/toxsci/kfm303

    Article  CAS  Google Scholar 

  • Brannen KC, Panzica-Kelly JM, Danberry TL, Augustine-Rauch KA (2010) Development of a zebrafish embryo teratogenicity assay and quantitative prediction model. Birth Defects Res B Dev Reprod Toxicol 89:66–77. doi:10.1002/bdrb.20223

    Article  CAS  Google Scholar 

  • Braunbeck T, Boettcher M, Hollert H et al (2005) Towards an alternative for the acute fish LC(50) test in chemical assessment: the fish embryo toxicity test goes multi-species—an update. ALTEX 22:87–102

    Google Scholar 

  • Burlinson B, Tice RR, Speit G et al (2007) Fourth International Workgroup on Genotoxicity testing: results of the in vivo Comet assay workgroup. Mutat Res 627:31–35. doi:10.1016/j.mrgentox.2006.08.011

    Article  CAS  Google Scholar 

  • Calabrese EJ, Blain RB (2011) The hormesis database: the occurrence of hormetic dose responses in the toxicological literature. Regul Toxicol Pharmacol RTP 61:73–81. doi:10.1016/j.yrtph.2011.06.003

    Article  CAS  Google Scholar 

  • Carls MG, Holland L, Larsen M et al (2008) Fish embryos are damaged by dissolved PAHs, not oil particles. Aquat Toxicol Amst Neth 88:121–127. doi:10.1016/j.aquatox.2008.03.014

    Article  CAS  Google Scholar 

  • Cole LK, Ross LS (2001) Apoptosis in the developing zebrafish embryo. Dev Biol 240:123–142. doi:10.1006/dbio.2001.0432

    Article  CAS  Google Scholar 

  • Collins AR, Oscoz AA, Brunborg G et al (2008) The comet assay: topical issues. Mutagenesis 23:143–151. doi:10.1093/mutage/gem051

    Article  CAS  Google Scholar 

  • David RM, Jones HS, Panter GH et al (2012) Interference with xenobiotic metabolic activity by the commonly used vehicle solvents dimethylsulfoxide and methanol in zebrafish (Danio rerio) larvae but not Daphnia magna. Chemosphere 88:912–917. doi:10.1016/j.chemosphere.2012.03.018

    Article  CAS  Google Scholar 

  • Doak SH, Jenkins GJS, Johnson GE et al (2007) Mechanistic influences for mutation induction curves after exposure to DNA-reactive carcinogens. Cancer Res 67:3904–3911. doi:10.1158/0008-5472.CAN-06-4061

    Article  CAS  Google Scholar 

  • EFSA (2009) Guidance of the scientific comitee on a request from EFSA on the use of the benchmark dose approach in risk assessment. EFSA J 1150:1–72

    Google Scholar 

  • Feiler U, Höss S, Ahlf W et al (2013) Sediment contact tests as a tool for the assessment of sediment quality in German waters. Environ Toxicol Chem SETAC 32:144–155. doi:10.1002/etc.2024

    Article  CAS  Google Scholar 

  • Fischer S, Klüver N, Burkhardt-Medicke K et al (2013) Abcb4 acts as multixenobiotic transporter and active barrier against chemical uptake in zebrafish (Danio rerio) embryos. BMC Biol 11:69. doi:10.1186/1741-7007-11-69

    Article  Google Scholar 

  • Fleming CR, Di Giulio RT (2011) The role of CYP1A inhibition in the embryotoxic interactions between hypoxia and polycyclic aromatic hydrocarbons (PAHs) and PAH mixtures in zebrafish (Danio rerio). Ecotoxicol Lond Engl 20:1300–1314. doi:10.1007/s10646-011-0686-1

    Article  CAS  Google Scholar 

  • Franco R, Panayiotidis MI (2010) Cell death or survival: the double-edged sword of environmental and occupational toxicity. Chem Biol Interact 188:265–266. doi:10.1016/j.cbi.2010.06.002

    Article  CAS  Google Scholar 

  • Garner LVT, Di Giulio RT (2012) Glutathione transferase pi class 2 (GSTp2) protects against the cardiac deformities caused by exposure to PAHs but not PCB-126 in zebrafish embryos. Comp Biochem Physiol Toxicol Pharmacol CBP 155:573–579. doi:10.1016/j.cbpc.2012.01.007

    Article  CAS  Google Scholar 

  • Gollapudi BB, Johnson GE, Hernandez LG et al (2013) Quantitative approaches for assessing dose–response relationships in genetic toxicology studies. Environ Mol Mutagen 54:8–18. doi:10.1002/em.21727

    Article  CAS  Google Scholar 

  • Grummt T, Kuckelkorn J, Bahlmann A et al (2013) Tox-Box: securing drops of life—an enhanced health-related approach for risk assessment of drinking water in Germany. Environ Sci Eur 25:27. doi:10.1186/2190-4715-25-27

    Article  Google Scholar 

  • Häfeli N, Schwartz P, Burkhardt-Holm P (2011) Embryotoxic and genotoxic potential of sewage system biofilm and river sediment in the catchment area of a sewage treatment plant in Switzerland. Ecotoxicol Environ Saf 74:1271–1279. doi:10.1016/j.ecoenv.2011.03.008

    Article  Google Scholar 

  • Hafner C, Gartiser S, Garcia-Käufer M et al. (2015) Investigations on sediment toxicity of German rivers applying a standardized bioassay battery. DanTox Project. (in this issue)

  • Hartmann A, Schumacher M, Plappert-Helbig U et al (2004) Use of the alkaline in vivo Comet assay for mechanistic genotoxicity investigations. Mutagenesis 19:51–59

    Article  CAS  Google Scholar 

  • Helma C, Uhl M (2000) A public domain image-analysis program for the single-cell gel-electrophoresis (comet) assay. Mutat Res 466:9–15

    Article  CAS  Google Scholar 

  • Hermsen SAB, van den Brandhof E-J, van der Ven LTM, Piersma AH (2011) Relative embryotoxicity of two classes of chemicals in a modified zebrafish embryotoxicity test and comparison with their in vivo potencies. Toxicol Vitro Int J Publ Assoc BIBRA 25:745–753. doi:10.1016/j.tiv.2011.01.005

    Article  CAS  Google Scholar 

  • Hernandez LG, Slob W, van SH, van BJ (2011) Can carcinogenic potency be predicted from in vivo genotoxicity data?: a meta-analysis of historical data. Env Mol Mutagen 52:518–528. doi:10.1002/em.20651

  • Hollert H, Keiter S, König N et al (2003) A new sediment contact assay to assess particle-bound pollutants using zebrafish (danio rerio) embryos. J Soils Sediments 3:197–207. doi:10.1065/jss2003.09.085

    Article  Google Scholar 

  • Hornung MW, Cook PM, Fitzsimmons PN et al (2007) Tissue distribution and metabolism of benzo[a]pyrene in embryonic and larval medaka (Oryzias latipes). Toxicol Sci Off J Soc Toxicol 100:393–405. doi:10.1093/toxsci/kfm231

    Article  CAS  Google Scholar 

  • Höss S, Ahlf W, Fahnenstich C et al (2010) Variability of sediment-contact tests in freshwater sediments with low-level anthropogenic contamination—determination of toxicity thresholds. Environ Pollut 158:2999–3010. doi:10.1016/j.envpol.2010.05.013

  • Incardona JP, Carls MG, Teraoka H et al (2005) Aryl hydrocarbon receptor-independent toxicity of weathered crude oil during fish development. Environ Health Perspect 113:1755–1762

    Article  CAS  Google Scholar 

  • Incardona JP, Linbo TL, Scholz NL (2011) Cardiac toxicity of 5-ring polycyclic aromatic hydrocarbons is differentially dependent on the aryl hydrocarbon receptor 2 isoform during zebrafish development. Toxicol Appl Pharmacol 257:242–249. doi:10.1016/j.taap.2011.09.010

    Article  CAS  Google Scholar 

  • ISO 7346–3 (1996) ISO 7346–3:1996 - Water quality -- Determination of the acute lethal toxicity of substances to a freshwater fish [Brachydanio rerio Hamilton-Buchanan (Teleostei, Cyprinidae)] -- Part 3: flow-through method. http://www.iso.org/iso/catalogue_detail.htm?csnumber=14030

  • Kais B, Schneider KE, Keiter S et al (2013) DMSO modifies the permeability of the zebrafish (Danio rerio) chorion—implications for the fish embryo test (FET). Aquat Toxicol Amst Neth 140–141:229–238. doi:10.1016/j.aquatox.2013.05.022

    Article  Google Scholar 

  • Keiter S, Rastall A, Kosmehl T et al (2006) Ecotoxicological assessment of sediment, suspended matter and water samples in the upper Danube River. A pilot study in search for the causes for the decline of fish catches. Environ Sci Pollut Res Int 13:308–319

    Article  CAS  Google Scholar 

  • Keiter S, Peddinghaus S, Feiler U et al (2010) DanTox—a novel joint research project using zebrafish (Danio rerio) to identify specific toxicity and molecular modes of action of sediment-bound pollutants. J Soils Sediments 10:714–717. doi:10.1007/s11368-010-0221-7

    Article  CAS  Google Scholar 

  • Kimmel CB, Ballard WW, Kimmel SR et al (1995) Stages of embryonic development of the zebrafish. Dev Dyn 203:253–310

    Article  CAS  Google Scholar 

  • Kosmehl T, Hallare AV, Reifferscheid G et al (2006) A novel contact assay for testing genotoxicity of chemicals and whole sediments in zebrafish embryos. Env Toxicol Chem 25:2097–2106

    Article  CAS  Google Scholar 

  • Kosmehl T, Krebs F, Manz W et al (2007) Differentiation between bioavailable and total hazard potential of sediment-induced DNA fragmentation as measured by the comet assay with Zebrafish embryos. J Soils Sediments 7:377–387. doi:10.1065/jss2007.11.261

    Article  CAS  Google Scholar 

  • Lee KJ, Nallathamby PD, Browning LM et al (2007) In vivo imaging of transport and biocompatibility of single silver nanoparticles in early development of zebrafish embryos. ACS Nano 1:133–143. doi:10.1021/nn700048y

    Article  CAS  Google Scholar 

  • Lin S, Zhao Y, Nel AE, Lin S (2013) Zebrafish: an in vivo model for nano EHS studies. Small 9:1608–1618

    Article  CAS  Google Scholar 

  • Lo KH, Hui MNY, Yu RMK et al (2011) Hypoxia impairs primordial germ cell migration in zebrafish (Danio rerio) embryos. PLoS One 6:e24540. doi:10.1371/journal.pone.0024540

    Article  CAS  Google Scholar 

  • McElroy A, Clark C, Duffy T et al (2012) Interactions between hypoxia and sewage-derived contaminants on gene expression in fish embryos. Aquat Toxicol Amst Neth 108:60–69. doi:10.1016/j.aquatox.2011.10.017

    Article  CAS  Google Scholar 

  • Meyer J, Di Giulio R (2002) Patterns of heritability of decreased EROD activity and resistance to PCB 126-induced teratogenesis in laboratory-reared offspring of killifish (Fundulus heteroclitus) from a creosote-contaminated site in the Elizabeth River, VA, USA. Mar Environ Res 54:621–626

    Article  CAS  Google Scholar 

  • Nagel R (2002) DarT: the embryo test with the Zebrafish Danio rerio—a general model in ecotoxicology and toxicology. ALTEX 19(Suppl 1):38–48

    Google Scholar 

  • OECD (2004) Test no. 218: sediment-water chironomid toxicity using spiked sediment. OECD Publishing. doi:10.1787/9789264070264-en

  • OECD (2014) Test no. 489: in vivo mammalian alkaline comet assay. OECD Publishing. doi:10.1787/9789264091016-en

  • Rocha PS, Bernecker C, Strecker R, et al (2011) Sediment-contact fish embryo toxicity assay with Danio rerio to assess particle-bound pollutants in the Tietê River Basin (São Paulo, Brazil). Ecotoxicol Environ Saf 74:1951–1959. doi:10.1016/j.ecoenv.2011.07.009

  • Schiwy S, Bräunig J, Alert H et al (2014) A novel contact assay for testing aryl hydrocarbon receptor (AhR)-mediated toxicity of chemicals and whole sediments in zebrafish (Danio rerio) embryos. Environ Sci Pollut Res Int. doi:10.1007/s11356-014-3185-0

    Google Scholar 

  • Scholz S, Fischer S, Gündel U, et al (2008) The zebrafish embryo model in environmental risk assessment--applications beyond acute toxicity testing. Environ Sci Pollut Res Int 15:394–404. doi:10.1007/s11356-008-0018-z

  • Seitz N, Böttcher M, Keiter S et al (2008) A novel statistical approach for the evaluation of comet assay data. Mutat Res 652:38–45. doi:10.1016/j.mrgentox.2007.12.004

    Article  CAS  Google Scholar 

  • Selderslaghs IWT, Blust R, Witters HE (2012) Feasibility study of the zebrafish assay as an alternative method to screen for developmental toxicity and embryotoxicity using a training set of 27 compounds. Reprod Toxicol Elmsford N 33:142–154. doi:10.1016/j.reprotox.2011.08.003

    Article  CAS  Google Scholar 

  • Sipes NS, Padilla S, Knudsen TB (2011) Zebrafish: as an integrative model for twenty-first century toxicity testing. Birth Defect Res Part C Embryo Today Rev 93:256–267. doi:10.1002/bdrc.20214

    Article  CAS  Google Scholar 

  • Strähle U, Scholz S, Geisler R et al (2012) Zebrafish embryos as an alternative to animal experiments—a commentary on the definition of the onset of protected life stages in animal welfare regulations. Reprod Toxicol Elmsford N 33:128–132. doi:10.1016/j.reprotox.2011.06.121

    Article  Google Scholar 

  • Strecker R, Seiler T-B, Hollert H, Braunbeck T (2011) Oxygen requirements of zebrafish (Danio rerio) embryos in embryo toxicity tests with environmental samples. Comp Biochem Physiol Toxicol Pharmacol CBP 153:318–327. doi:10.1016/j.cbpc.2010.12.002

    Article  Google Scholar 

  • Swenberg JA, Lu K, Moeller BC et al (2011) Endogenous versus exogenous DNA adducts: their role in carcinogenesis, epidemiology, and risk assessment. Toxicol Sci Off J Soc Toxicol 120(Suppl 1):S130–S145. doi:10.1093/toxsci/kfq371

    Article  CAS  Google Scholar 

  • Teixidó E, Piqué E, Gómez-Catalán J, Llobet JM (2013) Assessment of developmental delay in the zebrafish embryo teratogenicity assay. Toxicol Vitro Int J Publ Assoc BIBRA 27:469–478. doi:10.1016/j.tiv.2012.07.010

    Article  Google Scholar 

  • Tice RR, Agurell E, Anderson D et al (2000) Single cell gel/comet assay: guidelines for in vitro and in vivo genetic toxicology testing. Environ Mol Mutagen 35:206–221

    Article  CAS  Google Scholar 

  • Tuikka AI, Schmitt C, Hoss S et al (2011) Toxicity assessment of sediments from three European river basins using a sediment contact test battery. Ecotoxicol Environ Saf 74:123–131

    Article  CAS  Google Scholar 

  • Vasquez MZ (2010) Combining the in vivo comet and micronucleus assays: a practical approach to genotoxicity testing and data interpretation. Mutagenesis 25:187–199. doi:10.1093/mutage/gep060

    Article  CAS  Google Scholar 

  • Vasquez MZ (2012) Recommendations for safety testing with the in vivo comet assay. Mutat Res 747:142–156. doi:10.1016/j.mrgentox.2012.05.002

    Article  CAS  Google Scholar 

  • Vincze K, Graf K, Scheil V et al (2014) Embryotoxic and proteotoxic effects of water and sediment from the Neckar River (Southern Germany) to zebrafish (Danio rerio) embryos. Environ Sci Eur 26:3. doi:10.1186/2190-4715-26-3

    Article  Google Scholar 

  • Weigt S, Huebler N, Strecker R et al (2011) Zebrafish (Danio rerio) embryos as a model for testing proteratogens. Toxicology 281:25–36. doi:10.1016/j.tox.2011.01.004

    Article  CAS  Google Scholar 

  • Wills LP, Zhu S, Willett KL, Di Giulio RT (2009) Effect of CYP1A inhibition on the biotransformation of benzo[a]pyrene in two populations of Fundulus heteroclitus with different exposure histories. Aquat Toxicol Amst Neth 92:195–201. doi:10.1016/j.aquatox.2009.01.009

    Article  CAS  Google Scholar 

  • Wölz J, Brack W, Moehlenkamp C et al (2010) Effect-directed analysis of Ah receptor-mediated activities caused by PAHs in suspended particulate matter sampled in flood events. Sci Total Environ 408:3327–3333. doi:10.1016/j.scitotenv.2010.03.029

    Article  Google Scholar 

  • Woo S, Kim S, Yum S et al (2006) Comet assay for the detection of genotoxicity in blood cells of flounder (Paralichthys olivaceus) exposed to sediments and polycyclic aromatic hydrocarbons. Mar Pollut Bull 52:1768–1775. doi:10.1016/j.marpolbul.2006.08.027

    Article  CAS  Google Scholar 

  • Yu KN, Tung MMT, Choi VWY, Cheng SH (2012) Alpha radiation exposure decreases apoptotic cells in zebrafish embryos subsequently exposed to the chemical stressor, Cd. Environ Sci Pollut Res Int 19:3831–3839. doi:10.1007/s11356-012-1032-8

    Article  CAS  Google Scholar 

  • Zielke H, Seiler T-B, Niebergall S et al (2011) The impact of extraction methodologies on the toxicity of sediments in the zebrafish (Danio rerio) embryo test. J Soils Sediments 11:352–363. doi:10.1007/s11368-010-0317-0

    Article  CAS  Google Scholar 

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Acknowledgments

The present study was part of the joint research project DanTox (grant nos. 02WU1053), funded by the German Federal Ministry of Education and Research (BMBF). We also wish to thank Prof. W. Driever and S. Götter (Dept. of Developmental Biology, University of Freiburg, Germany) for kindly providing us with wild-type strains and breeding knowledge.

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Author notes

  1. S. Keiter

    Present address: Man-Technology-Environment Research Centre, School of Science and Technology, Örebro University, 70182, Örebro, Sweden

Authors and Affiliations

  1. Hydrotox GmbH, Bötzingerstr. 109, 79098, Freiburg, Germany

    M. Garcia-Käufer, S. Gartiser & C. Hafner

  2. Department of Ecosystem Analysis, Institute for Environmental Research, ABBT–Aachen Biology and Biotechnology, RWTH Aachen University, Worringerweg 1, 52074, Aachen, Germany

    M. Garcia-Käufer, S. Schiwy, S. Keiter & H. Hollert

  3. Center for Complementary Medicine, Department of Environmental Health Sciences, University Medical Centre Freiburg, Breisacherstr. 115b, 79106, Freiburg, Germany

    M. Garcia-Käufer & C. Gründemann

  4. School of Environment, Nanjing University, Nanjing, China

    H. Hollert

  5. Key Laboratory of Yangtze River Environment of Education Ministry of China, College of Environmental Science and Engineering, Tongji University, Shanghai, China

    H. Hollert

  6. College of Resources and Environmental Science, Chongqing University, Chongqing, China

    H. Hollert

  7. Department of Environmental Health Sciences, University Medical Centre Freiburg, Breisacherstr. 115b, 79106, Freiburg, Germany

    M. Garcia-Käufer

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  1. M. Garcia-Käufer
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Correspondence to M. Garcia-Käufer.

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Garcia-Käufer, M., Gartiser, S., Hafner, C. et al. Genotoxic and teratogenic effect of freshwater sediment samples from the Rhine and Elbe River (Germany) in zebrafish embryo using a multi-endpoint testing strategy. Environ Sci Pollut Res 22, 16341–16357 (2015). https://doi.org/10.1007/s11356-014-3894-4

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