Journal of Soils and Sediments

, Volume 11, Issue 2, pp 352–363 | Cite as

The impact of extraction methodologies on the toxicity of sediments in the zebrafish (Danio rerio) embryo test

  • Hanno Zielke
  • Thomas-Benjamin Seiler
  • Sabine Niebergall
  • Erik Leist
  • Markus Brinkmann
  • Denise Spira
  • Georg Streck
  • Werner Brack
  • Ute Feiler
  • Thomas Braunbeck
  • Henner Hollert
SEDIMENTS, SEC 1 • SEDIMENT QUALITY AND IMPACT ASSESSMENT • RESEARCH ARTICLE

Abstract

Purpose

Traditionally, methods for sediment extractions are characterised using chemical analyses. However, in order to evaluate sediment extracts with regard to biological effects and, thus, bioaccessibility, extraction methods have to be compared to effect data obtained from experiments with in situ exposure scenarios, i.e., sediment contact tests. This study compares four extraction methods and sediment contact test data from a previous project with respect to predictive power in the fish embryo test with zebrafish (Danio rerio).

Materials and methods

A natural and an artificial sediment spiked with a mixture of six organic pollutants (2,4-dinitrophenol, diuron, fluoranthene, nonylphenol, parathion and pentachlorophenol) were extracted using (a) membrane dialysis extraction (MDE), (b) a Soxhlet procedure, (c) hydroxypropyl-β-cyclodextrin (HPCD) or (d) Tenax®-TA. Whereas the former two are regarded being exhaustive with respect to non-covalently bound contaminants, the latter two are considered to predict bioaccessibility. Resulting extracts were tested in the fish embryo assay with D. rerio for embryotoxic and teratogenic potential.

Results and discussion

Mortalities caused by organic extracts from Soxhlet extraction and MDE were high. However, HPCD extracts turned out to be at least as effective as extracts obtained with these two methods. One possible reason might be short ageing time of the spiked sediments. Only Tenax®-TA extracts gave results comparable to the sediment contact assay for natural sediment, but revealed low reproducibility. Significant differences between natural and artificial sediment were found for extracts obtained with techniques using native (i.e., non-freeze-dried) sediments, i.e., HPCD and Tenax®-TA. In contrast, MDE and Soxhlet extracts used freeze-dried sediment and did not differentiate between natural and artificial sediment. Therefore, freeze-drying has likely altered and equalised sediment properties that influence accessibility, such as composition of bacterial communities and organic matter quality.

Conclusions

Four extraction methods were successfully characterised with respect to their stringency and predictiveness for bioaccessibility. MDE was confirmed as an alternative to Soxhlet extraction. High mortalities induced by HPCD extracts underline the need to include ageing into consideration when assessing sediments. Although Tenax®-TA may basically be used to predict bioaccessibility in the fish embryo test, the high variability observed warrants further investigation of the relation between effect and extractability. Apparently, freeze-drying can severely affect sediment properties, potentially eliminating individual properties of natural sediments.

Keywords

Cyclodextrin Extraction Fish embryo test Membrane dialysis extraction Tenax Sediment Soxhlet Zebrafish 

References

  1. Alexander M (1995) How toxic are toxic chemicals in soil? Environ Sci Technol 29:2713–2717CrossRefGoogle Scholar
  2. Alexander M (2000) Aging, bioavailability, and overestimation of risk from environmental pollutants. Environ Sci Technol 34:4259–4265CrossRefGoogle Scholar
  3. Andersson E, Rotander A, von Kronhelm T, Berggren A, Ivarsson P, Hollert H, Engwall M (2009) AhR agonist and genotoxicant bioavailability in a PAH-contaminated soil undergoing biological treatment. Environ Sci Poll Res 16:521–530CrossRefGoogle Scholar
  4. Arditsoglou A, Voutsa D (2008) Determination of phenolic and steroid endocrine disrupting compounds in environmental matrices. Environ Sci Poll Res 15:228–236CrossRefGoogle Scholar
  5. Bjorklund E, Holst C, Anklam E (2002) Fast extraction, clean-up and detection methods for the rapid analysis and screening of seven indicator PCBs in food matrices. Trends Anal Chem 21:39–52CrossRefGoogle Scholar
  6. Brack W (2003) Effect-directed analysis: a promising tool for the identification of organic toxicants in complex mixtures? Anal Bioanal Chem 377:397–407CrossRefGoogle Scholar
  7. Brack W, Schirmer K, Erdinger L, Hollert H (2005) Effect-directed analysis of mutagens and ethoxyresorufin-O-deethylase inducers in aquatic sediments. Environ Toxicol Chem 24:2445–2458CrossRefGoogle Scholar
  8. Brack W, Bandow N, Schwab K, Schulze T, Streck G (2009) Bioavailability in effect-directed analysis of organic toxicants in sediments. Trends Anal Chem 28:543–549CrossRefGoogle Scholar
  9. Braunbeck T, Boettcher M, Hollert H, Kosmehl T, Lammer E, Leist E, Rudolf M, Seitz N (2005) Towards an alternative for the acute fish LC50 test in chemical assessment: The fish embryo toxicity test goes multi-species—an update. Altex 22:87–102Google Scholar
  10. Brils J et al (2007) Sediment management: An essential element of river basin management plans. J Soils Sediments 7:117–132CrossRefGoogle Scholar
  11. Burton GA (1991) Assessing the toxicity of fresh-water sediments. Environ Toxicol Chem 10:1585–1627CrossRefGoogle Scholar
  12. Carroll KM, Harkness MR, Bracco AA, Balcarcel RR (1994) Application of a Permeant Polymer Diffusional Model to the Desorption of Polychlorinated-Biphenyls from Hudson River Sediments. Environ Sci Technol 28:253–258CrossRefGoogle Scholar
  13. Chung N, Alexander M (1999) Effect of concentration on sequestration and bioavailability of two polycyclic aromatic hydrocarbons. Environ Sci Technol 33:3605–3608CrossRefGoogle Scholar
  14. Cornelissen G, Van Noort PCM, Govers HAJ (1997) Desorption kinetics of chlorobenzenes, polycyclic aromatic hydrocarbons, and polychlorinated biphenyls: Sediment extraction with Tenax® and effects of contact time and solute hydrophobicity. Environ Toxicol Chem 16:1351–1357CrossRefGoogle Scholar
  15. Cornelissen G, Rigterink H, Ten Hulscher DEM, Vrind BA, Van Noort PCM (2001) A simple tenax® extraction method to determine the availability of sediment-sorbed organic compounds. Environ Toxicol Chem 20:706–711CrossRefGoogle Scholar
  16. Cuypers C, Clemens R, Grotenhuis T, Rulkens W (2001) Prediction of petroleum hydrocarbon bioavailability in contaminated soils and sediments. Soil Sediment Contam 10:459–482CrossRefGoogle Scholar
  17. Cuypers C, Pancras T, Grotenhuis T, Rulkens W (2002) The estimation of PAH bioavailability in contaminated sediments using hydroxypropyl-ß-cyclodextrin and Triton X-100 extraction techniques. Chemosphere 46:1235–1245CrossRefGoogle Scholar
  18. Cuypers H, Grotenhuis T, Joziasse J, Rulkens W (2000) Rapid persulfate oxidation predicts PAH bioavailability in soils and sediments. Environ Sci Technol 34:2057–2063CrossRefGoogle Scholar
  19. De la Cal A, Eljarrat E, Grotenhuis T, Barcelo D (2008) Tenax (R) extraction as a tool to evaluate the availability of polybrominated diphenyl ethers, DDT, and DDT metabolites in sediments. Environ Toxicol Chem 27:1250–1256CrossRefGoogle Scholar
  20. DIN (2001) DIN 38415-6: Deutsche Einheitsverfahren zur Wasser-, Abwasser- und Schlammuntersuchung: Suborganismische Testverfahren (Gruppe T) Teil 6: Giftigkeit gegenüber Fischen: Bestimmung der nicht akut giftigen Wirkung von Abwasser auf die Entwicklung von Fischeiern über Verdünnungsstufen (T 6). Deutsches Institut für Normung e. V., Berlin, 14 S.Google Scholar
  21. EP/EC (2008) Directive 2008/105/EC of the european parliament and of the council of 16 December 2008 on environmental quality standards in the field of water policy, amending and subsequently repealing Council Directives 82/176/EEC, 83/513/EEC, 84/156/EEC, 84/491/EEC, 86/280/EEC and amending Directive 2000/60/EC of the European Parliament and of the Council. http://eur-lex.europa.eu/LexUriServ/LexUriServ.do?uri=OJ:L:2008:348:0084:0097:EN:PDF
  22. Ehlers GAC, Loibner AP (2006) Linking organic pollutant (bio)availability with geosorbent properties and biomimetic methodology: a review of geosorbent characterisation and (bio)availability prediction. Environ Pollut 141:494–512CrossRefGoogle Scholar
  23. Feiler U, Kirchesch I, Heininger P (2004) A new plant-based bioassay for aquatic sediments. J Soils Sediments 4:261–266CrossRefGoogle Scholar
  24. Feiler U, Ahlf W, Hoess S, Hollert H, Neumann-Hensel H, Meller M, Weber J, Heininger P (2005) The SeKT Joint Research Project: Definition of reference conditions, control sediments and toxicity thresholds for limnic sediment contact tests. Environ Sci Poll Res 12:257–258CrossRefGoogle Scholar
  25. Feiler U, Ahlf W, Fahnenstich C, Gilberg D, Hammers-Wirtz M, Höss S, Hollert H, Melbye K, Meller M, Neumann-Hensel H, Ratte HT, Seiler TB, Spira D, Weber J, Heininger (2009a) Definition von Referenzbedingungen, Kontrollsedimenten und Toxizitätsschwellenwerten für limnische Sedimentkontakttetst (SeKT) - Final project report BfG1614, Federal Institute of Hydrology, Koblenz, Germany, ISBN 978-3-940247-01-8, 251 p.Google Scholar
  26. Feiler U, Claus E, Spira D, Heininger P (2009b) Aquatic plant bioassays used in the assessment of sediment quality. Umweltwiss Schadst Forsch 21:264–266CrossRefGoogle Scholar
  27. Fleming RJ, Holmes D, Nixon SJ (1998) Toxicity of permethrin to Chironomus riparius in artificial and natural sediments. Environ Toxicol Chem 17:1332–1337Google Scholar
  28. Förstner U (2009) Sediments and priority substances in river basins. J Soils Sediments 9:89–93CrossRefGoogle Scholar
  29. Ghosh U, Gillette JS, Luthy RG, Zare RN (2000) Microscale location, characterization, and association of polycyclic aromatic hydrocarbons on harbor sediment particles. Environ Sci Technol 34:1729–1736CrossRefGoogle Scholar
  30. Goedkoop W, Widenfalk A, Haglund AL, Steger K, Bertilsson S (2005) Microbial characterization of artificial sediment and comparisons with natural sediments—implications for toxicity testing. Environ Toxicol Chem 24:2725–2733CrossRefGoogle Scholar
  31. Hallare AV, Kosmehl T, Schulze T, Hollert H, Kohler HR, Triebskorn R (2005) Assessing contamination levels of Laguna Lake sediments (Philippines) using a contact assay with zebrafish (Danio rerio) embryos. Sci Total Environ 347:254–271CrossRefGoogle Scholar
  32. Hallare AV, Seiler TB, Hollert H (2010) The versatile, changing, and advancing roles of fish in sediment toxicity assessment—a review. J Soils Sediments. doi:10.1007/s11368-010-0302-7 Google Scholar
  33. Hawthorne SB, Grabanski CB, Hageman KJ, Miller DJ (1998) Simple method for estimating polychlorinated biphenyl concentrations on soils and sediments using subcritical water extraction coupled with solid-phase microextraction. J Chromatogr A 814:151–160CrossRefGoogle Scholar
  34. Heise S, Ahlf W (2005) A new microbial contact assay for marine sediments—dedicated to Prof. Dr. Ulrich Forstner on his 65th birthday. J Soils Sediments 5:9–15CrossRefGoogle Scholar
  35. Hickman ZA, Reid BJ (2005) Towards a more appropriate water based extraction for the assessment of organic contaminant availability. Environ Pollut 138:299–306CrossRefGoogle Scholar
  36. Hollert H, Durr M, Erdinger L, Braunbeck T (2000) Cytotoxicity of settling particulate matter and sediments of the Neckar River (Germany) during a winter flood. Environ Toxicol Chem 19:528–534CrossRefGoogle Scholar
  37. Hollert H, Keiter S, König N, Rudolf M, Ulrich M, Braunbeck T (2003) A new sediment contact assay to assess particle-bound pollutants using zebrafish (Danio rerio) embryos. J Soils Sediments 3:197–207CrossRefGoogle Scholar
  38. Hollert H, Seiler TB, Blaha L, Young AL (2007) Multiple stressors for the environment: present and future challenges and perspectives. Environ Sci Poll Res 14:222–222CrossRefGoogle Scholar
  39. Hollert H, Ernst M, Ahlf W, Duerr M, Erdinger L, Grund S, Keiter S, Kosmehl T, Seiler T-B, Woelz J, Braunbeck T (2009) Strategies for assessing sediment toxicity—a review. Umweltwiss Schadstoff Forsch 21:160–176CrossRefGoogle Scholar
  40. Höss S, Ahlf W, Fahnenstich C, Gilberg D, Hollert H, Melbye K, Meller M, Hammers-Wirtz M, Heininger P, Neumann-Hensel H, Ottermanns R, Ratte HT, Seiler TB, Spira D, Weber J, Feiler U (2010) Variability of freshwater sediment contact tests in sediments with low anthropogenic contamination—determination of toxicity thresholds. Environ Pollut. doi:10.1016/j.envpol.2010.05.013 Google Scholar
  41. Huang WL, Ping PA, Yu ZQ, Fu HM (2003) Effects of organic matter heterogeneity on sorption and desorption of organic contaminants by soils and sediments. Appl Geochem 18:955–972CrossRefGoogle Scholar
  42. Huckins JN, Tubergen MW, Lebo JA, Gale RW, Schwartz TR (1990) Polymeric film dialysis in organic-solvent media for cleanup of organic contaminants. J Assoc Off Ana Chem 73:290–293Google Scholar
  43. ISO (International Organization for Standardization) (1996) ISO 7346/3: 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. Iso GuidelineGoogle Scholar
  44. Karlsson J, Sundberg H, Akerman G, Grunder K, Eklund B, Breitholtz M (2008) Hazard identification of contaminated sites—ranking potential toxicity of organic sediment extracts in crustacean and fish. J Soils Sediments 8:263–274CrossRefGoogle Scholar
  45. Kosmehl T, Krebs F, Manz W, Braunbeck T, Hollert H (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 7:377–387CrossRefGoogle Scholar
  46. Lammer E, Carr GJ, Wendler K, Rawlings JM, Belanger SE, Braunbeck T (2009) Is the fish embryo toxicity test (FET) with the zebrafish (Danio rerio) a potential alternative for the fish acute toxicity test? Comp Biochem Phys C 149:196–209Google Scholar
  47. Luo JP, Ma M, Liu C, Zha JM, Wang ZJ (2009) Impacts of particulate organic carbon and dissolved organic carbon on removal of polycyclic aromatic hydrocarbons, organochlorine pesticides, and nonylphenols in a wetland. J Soils Sediments 9:180–187CrossRefGoogle Scholar
  48. Luque de Castro MD, Garcia-Ayuso LE (1998) Soxhlet extraction of solid materials: an outdated technique with a promising innovative future. Anal Chim Acta 369:1–10CrossRefGoogle Scholar
  49. Macrae JD, Hall KJ (1998) Comparison of methods used to determine the availability of polycyclic aromatic hydrocarbons in marine sediment. Environ Sci Technol 32:3809–3815CrossRefGoogle Scholar
  50. Marklevitz SAC, Almeida E, Flemming J, Hellou J (2008) Determining the bioavailability of contaminants and assessing the quality of sediments. J Soils Sediments 8:86–91CrossRefGoogle Scholar
  51. Moermond CTA, Roessink I, Jonker MTO, Meijer T, Koelmans AA (2007) Impact of polychlorinated biphenyl and polycyclic aromatic hydrocarbon sequestration in sediment on bioaccumulation in aquatic food webs. Environ Toxicol Chem 26:607–615CrossRefGoogle Scholar
  52. Morrison DE, Robertson BK, Alexander M (2000) Bioavailability to earthworms of aged DDT, DDE, DDD, and dieldrin in soil. Environ Sci Technol 34:709–713CrossRefGoogle Scholar
  53. Nagel R (2002) DarT: The embryo test with the zebrafish Danio rerio—a general model in ecotoxicology and toxicology. Altex 19:38–48Google Scholar
  54. Nam K, Chung N, Alexander M (1998) Relationship between organic matter content of soil and the sequestration of phenanthrene. Environ Sci Technol 32:3785–3788CrossRefGoogle Scholar
  55. Neumann-Hensel H, Melbye K (2006) Optimisation of the solid-contact test with Artbrobacter globiformis. J Soils Sediments 6:201–207CrossRefGoogle Scholar
  56. Northcott GL, Jones KC (2000) Experimental approaches and analytical techniques for determining organic compound bound residues in soil and sediment. Environ Pollut 108:19–43CrossRefGoogle Scholar
  57. OECD (2004) OECD guideline 218: sediment-water chironomid toxicity using spiked sediment. Organisation for Economic Cooperation and Development, BerlinGoogle Scholar
  58. Pane L, Giacco E, Corra C, Greco G, Mariottini GL, Varisco F, Faimali M (2008) Ecotoxicological evaluation of harbour sediments using marine organisms from different trophic levels. J Soils Sediments 8:74–79CrossRefGoogle Scholar
  59. Qiao M, Huang S, Wang Z (2008) Partitioning characteristics of PAHs between sediment and water in a shallow lake. J Soils Sediments 8:69–73CrossRefGoogle Scholar
  60. Ramos EU, Meijer SN, Vaes WHJ, Verhaar HJM, Hermens JLM (1998) Using solid-phase microextraction to determine partition coefficients to humic acids and bioavailable concentrations of hydrophobic chemicals. Environ Sci Technol 32:3430–3435CrossRefGoogle Scholar
  61. Reichenberg F, Mayer P (2006) Two complementary sides of bioavailability: accessibility and chemical activity of organic contaminants in sediments and soils. Environ Sci Technol 25:1239–1245Google Scholar
  62. Reid BJ, Stokes JD, Jones KC, Semple KT (2000) Nonexhaustive cyclodextrin-based extraction technique for the evaluation of PAH bioavailability. Environ Sci Technol 34:3174–3179CrossRefGoogle Scholar
  63. Santos FJ, Sarrion MN, Galceran MT (1997) Analysis of chlorobenzenes in soils by headspace solid-phase microextraction and gas chromatography-ion trap mass spectrometry. J Chromatogr A 771:181–189CrossRefGoogle Scholar
  64. Schwab K, Brack W (2007) Large volume tenax (R) extraction of the bioaccessible fraction of sediment-associated organic compounds for a subsequent effect-directed analysis. J Soils Sediments 7:178–186CrossRefGoogle Scholar
  65. Seiler TB (2010) Total or biomimetic extracts or direct contact exposure? - Comparative research towards a realistic ecotoxicological characterisation of sediments. PhD thesis, Ruprecht-Karls University Heidelberg, 340 p., http://archiv.ub.uni-heidelberg.de/volltextserver/volltexte/2010/11171/pdf/20100701_Dissertation_Seiler_veroeff.pdf
  66. Seiler TB, Rastall AC, Leist E, Erdinger L, Braunbeck T, Hollert H (2006) Membrane dialysis extraction (MDE): A novel approach for extracting toxicologically relevant hydrophobic organic compounds from soils and sediments for assessment in biotests. J Soils Sediments 6:20–29CrossRefGoogle Scholar
  67. Seiler TB, Schulze T, Hollert H (2008) The risk of altering soil and sediment samples upon extract preparation for analytical and bio-analytical investigations—a review. Anal Bioanal Chem 390:1975–1985CrossRefGoogle Scholar
  68. Semple KT, Doick KJ, Jones KC, Burauel P, Craven A, Harms H (2004) Defining bioavailability and bioaccessibility of contaminated soil and sediment is complicated. Environ Sci Technol 38:228A–231ACrossRefGoogle Scholar
  69. Smit MPJ, Grotenhuis T, Bruning H, Rulkens WH (2008) Desorption of dieldrin from field aged sediments: simulating flood events. J Soils Sediments 8:80–85CrossRefGoogle Scholar
  70. Sormunen AJ, Leppanen MT, Kukkonen JVK (2009) Examining the role of temperature and sediment-chemical contact time on desorption and bioavailability of sediment-associated tetrabromo diphenyl ether and benzo(a)pyrene. Ecotox Environ Safe 72:1234–1241CrossRefGoogle Scholar
  71. Stokes JD, Wilkinson A, Reid BJ, Jones KC, Semple KT (2005) Prediction of polycyclic aromatic hydrocarbon biodegradation in contaminated soils using an aqueous hydroxypropyl-ß-cyclodextrin extraction technique. Environ Toxicol Chem 24:1325–1330CrossRefGoogle Scholar
  72. Swindell AL, Reid BJ (2006) Comparison of selected non-exhaustive extraction techniques to assess PAH availability in dissimilar soils. Chemosphere 62:1126–1134CrossRefGoogle Scholar
  73. Tang J, Robertson BK, Alexander M (1999) Chemical-extraction methods to estimate bioavailability of DDT, DDE, and DDD in soil. Environ Sci Technol 33:4346–4351CrossRefGoogle Scholar
  74. Tang J, Liste HH, Alexander M (2002) Chemical assays of availability to earthworms of polycyclic aromatic hydrocarbons in soil. Chemosphere 48:35–42CrossRefGoogle Scholar
  75. Tang JX, Alexander M (1999) Mild extractability and bioavailability of polycyclic aromatic hydrocarbons in soil. Environ Toxicol Chem 18:2711–2714CrossRefGoogle Scholar
  76. Ten Hulscher TEM, Postma J, Den Besten PJ, Stroomberg GJ, Belfroid A, Wegener JW, Faber JH, Van Der Pol JJC, Jan Hendriks A, Van Noort PCM (2003) Tenax extraction mimics benthic and terrestrial bioavailability of organic compounds. Environ Toxicol Chem 22:2258–2265CrossRefGoogle Scholar
  77. Van der Heijden SA, Jonker MTO (2009) PAH bioavailability in field sediments: Comparing different methods for predicting in situ bioaccumulation. Environ Sci Technol 43:3757–3763CrossRefGoogle Scholar
  78. Verrhiest GJ, Cortes S, Clement B, Montuelle B (2002) Chemical and bacterial changes during laboratory conditioning of formulated and natural sediments. Chemosphere 46:961–974CrossRefGoogle Scholar
  79. Wölz J, Engwall M, Maletz S, Takner HO, van Bavel B, Kammann U, Klempt M, Weber R, Braunbeck T, Hollert H (2008) Changes in toxicity and Ah receptor agonist activity of suspended particulate matter during flood events at the rivers Neckar and Rhine—a mass balance approach using in vitro methods and chemical analysis. Environ Sci Poll Res 15:536–553CrossRefGoogle Scholar
  80. Wölz J, Cofalla C, Hudjetz S, Roger S, Brinkmann M, Schmidt B, Schaffer A, Kammann U, Lennartz G, Hecker M, Schuttrumpf H, Hollert H (2009) In search for the ecological and toxicological relevance of sediment re-mobilisation and transport during flood events. J Soils Sediments 9:1–5CrossRefGoogle Scholar
  81. You J, Landrum PF, Lydy MJ (2006) Comparison of chemical approaches for assessing bioavailability of sediment-associated contaminants. Environ Sci Technol 40:6348–6353CrossRefGoogle Scholar
  82. Zambonin CG, Catucci F, Palmisano F (1998) Solid phase microextraction coupled to gas chromatography-mass spectrometry for the determination of the adsorption coefficients of triazines in soil. Analyst 123:2825–2828CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Hanno Zielke
    • 1
  • Thomas-Benjamin Seiler
    • 1
  • Sabine Niebergall
    • 2
  • Erik Leist
    • 2
  • Markus Brinkmann
    • 1
  • Denise Spira
    • 3
  • Georg Streck
    • 4
  • Werner Brack
    • 4
  • Ute Feiler
    • 3
  • Thomas Braunbeck
    • 2
  • Henner Hollert
    • 1
  1. 1.Department of Ecosystem Analysis, Institute for Environmental Research (Biology V)RWTH Aachen UniversityAachenGermany
  2. 2.Aquatic Ecology and Toxicology Section, Department of ZoologyUniversity of HeidelbergHeidelbergGermany
  3. 3.Department Biochemistry and EcotoxicologyGerman Federal Institute of HydrologyKoblenzGermany
  4. 4.Department Effect-Directed AnalysisHelmholtz Center of Environmental Research—UFZLeipzigGermany

Personalised recommendations