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Evaluation of the bioaccumulation potential of alizarin red S in fish muscle tissue using the European eel as a model


For fish stock management and large-scale stocking programs, the chemical substance alizarin red S (ARS) is an important tool to mark fish permanently. Equally, for the IUCN red list species European eel (Anguilla anguilla), ARS is proven to be the most promising option for mass marking. ARS binds to calcified structures (i.e., bones and otoliths) and can be detected using a fluorescence microscope. Despite the frequent application of ARS, not only for eels but also for fish in general, until today, no study has evaluated its bioaccumulation potential. Therefore, the German Federal Risk Assessment Authority was unable to classify ARS as harmless because of a potential risk to consumers’ health. Using the technique of liquid chromatography mass spectrometry, an ARS detection protocol was developed and the bioaccumulation potential of ARS in European eel muscle tissue was estimated. A detection limit of 8.9 μg kg−1 could be reached by optimizing the detection method in fish muscle tissue. In the current study, 250 eels between 6 and 57 cm of total length have been analyzed for ARS between 0 day and 3 years after the marking process. The highest concentration of ARS (6056 μg kg−1) was observed immediately after marking in the smallest length class. Only 1 year after the marking procedure, the ARS concentration was below detection limit. A new method for ARS detection in fish muscle tissue, followed by utilization on marked eels, was able to show that the bioaccumulation of ARS in edible fish muscle was highly unlikely.

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  1. 1.

    Nielsen LA. Methods of marking fish and shellfish. Am Fish Soc Special Publ. 1992;23:1–208.

  2. 2.

    Beamish RJ, McFarlane GA. The forgotten requirement for age validation in fisheries biology. Trans Am Fish Soc. 1983;112(6):735–43.

  3. 3.

    Warren-Myers F, Ingram BA, Dempster T, Swearer SE. Enriched stable isotope marking of hatchery trout via immersion: a method to monitor restocking success. Fish Res. 2018;197:78–83.

  4. 4.

    Wickström H, Sjöberg NB. Traceability of stocked eels—the Swedish approach. Ecol Freshw Fish. 2014;23(1):33–9.

  5. 5.

    Marohn L, Jakob E, Hanel R. Implications of facultative catadromy in Anguilla anguilla. Does individual migratory behaviour influence eel spawner quality? J Sea Res. 2013;77:100–6.

  6. 6.

    ICES. Report of the Joint EIFAAC/ICES/GFCM Working Group on Eel (WGEEL). Poland: Gdansk; 2018. p. 5–12. 15:1–152. http://www.ices.dk/sites/pub/Publication%20Reports/Expert%20Group%20Report/acom/2018/WGEEL/wgeel_2018.pdf. Accessed 30 Apr 2019

  7. 7.

    EC. Council Regulation (EC) No 1100/2007 of 18 September 2007 establishing measures for the recovery of the stock of European eel. 2007. http://eur-lex.europa.eu/legal-content/en/TXT/?uri=CELEX%3A32007R1100. Accessed 30 Apr 2019.

  8. 8.

    Fladung E, Brämick U. Umsetzungsbericht 2018 zu den Aalbewirtschaftungsplänen der deutschen Länder 2008. 2018;1-62. https://wwwportal-fischereide/fileadmin/redaktion/dokumente/fischerei/Bund/Bestandsmanagement/Umsetzungsbericht_dt_AMP_2018pdf. Access 30 Apr 2019.

  9. 9.

    ICES. Report of the Workshop on Eel Stocking (WKSTOCKEEL). Northern Ireland: Toomebridge; 2016. p. 20–4. 21:1–75. http://ices.dk/sites/pub/Publication%20Reports/Expert%20Group%20Report/SSGEPD/2016/01%20WKSTOCKEEL%20-%20Report%20of%20the%20Workshop%20on%20Eel%20Stocking.pdf. Accessed 30 Apr 2019

  10. 10.

    Angelidis P, Pournara I, Photis G. Glass eels (Anguilla anguilla) growth in a recirculating system. Med Mar Sci. 2005;6(1):99–106.

  11. 11.

    Pedersen MI, Rasmussen GH. Yield per recruit from stocking two different sizes of eel (Anguilla anguilla) in the brackish Roskilde Fjord. ICES J Mar Sci. 2016;73(1):158–64.

  12. 12.

    Nielsen T, Prouzet P. Captured-based aquaculture of the wild European eel (Anguilla anguilla). FAO Fish Tech Pap. 2008;508:141–68.

  13. 13.

    ICES. Report of the Workshop on Age Reading of European and American Eel (WKAREA2). Bordeaux, France; 2011. p. 22–4. 43:1–31. http://www.google.de/url?sa=t&rct=j&q=&esrc=s&source=web&cd=1&cad=rja&uact=8&ved=2ahUKEwi8kaW8jIfiAhVQr6QKHZM0AXUQFjAAegQIAhAB&url=http%3A%2F%2Fwww.ices.dk%2Fsites%2Fpub%2FPublication%2520Reports%2FExpert%2520Group%2520Report%2Facom%2F2011%2FWKAREA2%2FWKAREA2%25202011.pdf&usg=AOvVaw0hNtWs5QXwdfBe6lly1toP. Accessed 06 May 2019

  14. 14.

    Kullmann B, Pohlmann JD, Freese M, Keth A, Wichmann L, Neukamm R, et al. Age-based stock assessment of the European eel (Anguilla anguilla) is heavily biased by stocking of unmarked farmed eels. Fish Res. 2018b;208:258–66.

  15. 15.

    Caraguel JM, Charrier F, Mazel V, Feunteun E. Mass marking of stocked European glass eels (Anguilla anguilla) with alizarin red S. Ecol Freshw Fish. 2015;24(3):435–42.

  16. 16.

    Kullmann B, Neukamm R, Thiel R. Mass-marking of farmed European eels (Anguilla anguilla (Linnaeus, 1758)) with alizarin red S. J Appl Ichthyol. 2017;33(5):914–7.

  17. 17.

    Kullmann B, Thiel R. The chemical marking of eels—state of the art and application perspectives. In: Coulson P, editor. Eels biology, monitoring, management and exploitation. Charlbury: 5 m; 2019. p. 172–87.

  18. 18.

    Abdulrahman S, Basavaiah K. Use of alizarin red S as a chromogenic agent for the colorimetric determination of dothiepin hydrochloride in pharmaceutical formulations. J Saudi Chem Soc. 2014;18(2):107–14.

  19. 19.

    Springsteen G, Wang B. Alizarin red S as a general optical reporter for studying the binding of boronic acids with carbohydrates. Chem Commun. 2001;17:1608–9.

  20. 20.

    Schendel T, Jung C, Lindtner O, Greiner M. Guidelines for uncertainty analysis: application of the respective documents of EFSA and BfR for exposure assessments. EFSA J 2018; https://efsa.onlinelibrary.wiley.com/doi/pdf/10.2903/sp.efsa.2018.EN-1472. Accessed 30 April 2019.

  21. 21.

    Simon J, Dörner H. Marking the European eel with oxytetracycline, alizarin red and coded wire tags: an evaluation of methods. J Fish Biol. 2005;67(5):1486–91.

  22. 22.

    Rashid-Doubell F, Horobin RW. Selection of fluorescent Golgi complex probes using structure-activity relationship models. In: Bach PH, Reynolds CH, Clark JM, Mottley J, Poole PL, editors. Biotechnology applications of microinjection, microscopic imaging, and fluorescence. Boston: Springer; 1993. p. 73–4.

  23. 23.

    Anastassiades M, Lehotay SJ, Stajnbaher D, Schenck FJ. Safety data sheet Carl Roth. 2017. https://www.carlroth.com/downloads/sdb/de/3/SDB_3586_DE_DE.pdf. Access 30 Apr 2019.

  24. 24.

    Larsson P, Hamrin S, Okla L. Fat content as a factor inducing migratory behavior in the eel (Anguilla anguilla L.) to the Sargasso Sea. Naturwissenschaften. 1990;77:488–90.

  25. 25.

    Ahlgren G, Blomqvist P, Boberg M, Gustafsson IB. Fatty acid content of the dorsal muscle—an indicator of fat quality in freshwater fish. J Fish Biol. 1994;45(1):131–57.

  26. 26.

    Dorow M, Schaarschmidt T. Besatz mit Glasaalen in Küstengewässern 2015. In: Fischerei und Fischmarkt in Mecklenburg-Vorpommern. 2015;1(15):64–65. https://www.lfvmv.de/download/zeitschrift/FF_1_2015.pdf. Accessed 30 Apr 2019.

  27. 27.

    Kullmann B, Hempel M, Thiel R. Chemical marking of European glass eels Anguilla anguilla with alizarin red S and in combination with strontium: in situ evaluation of short-term salinity effects on survival and efficient mass-marking. J Fish Biol. 2018a;92(1):203–13.

  28. 28.

    ICES. Workshop on Age Reading of European and American Eel (WKAREA). France: Bordeaux; 2009. p. 20–4. 48:1–66. http://www.ices.dk/sites/pub/Publication%20Reports/Expert%20Group%20Report/acom/2009/WKAREA/WKAREA%202009.pdf. Accessed 30 Apr 2019

  29. 29.

    Kullmann B, Thiel R. Bigger is better in eel stocking measures? Comparison of growth performance, body condition, and benefit-cost ratio of simultaneously stocked glass and farmed eels in a brackish fjord. Fish Res. 2018c;205:132–40.

  30. 30.

    Bonhommeau S, Blanke B, Tréguier A, Grima N, Rivot E, Vermard Y, et al. How fast can the European eel (Anguilla anguilla) larvae cross the Atlantic Ocean? Fish Oceanogr. 2009;18(6):371–85.

  31. 31.

    Bonhommeau S, Castonguay M, Rivot E, Sabatié LP. The duration of migration of Atlantic Anguilla larvae. Fish Fish. 2010;11(3):289–306.

  32. 32.

    Westerberg H, Pacariz S, Marohn L, Fagerström V, Wysujack K, Miller MJ, et al. Modeling the drift of European (Anguilla anguilla) and American (Anguilla rostrata) eel larvae during the year of spawning. Can J Fish Aquat Sci. 2017;00:1–11.

  33. 33.

    Fuh MR, Chia KJ. Determination of sulphonated azo dyes in food by ion-pair liquid chromatography with photodiode array and electrospray mass spectrometry detection. Talanta. 2002;56(4):663–71.

  34. 34.

    Souto C. Analysis of early synthetic dyes with HPLC-DAD-MS: an important database for analysis of colorants used in cultural heritage. 2010. https://run.unl.pt/bitstream/10362/5656/1/Souto_2010.pdf. Accessed 30 Apr 2019.

  35. 35.

    Wee SL, Ching TT, Subhash B. Process optimization studies for the dehydration of alcohol water system by inorganic membrane based pervaporation separation using design of experiment (DoE). Sep Purif Technol. 2010;71(2):192–9.

  36. 36.

    DIN 32645:2008-11. Chemische Analytik - Nachweis-, Erfassungs- und Bestimmungsgrenze unter Wiederholbedingungen - Begriffe. Auswertung: Verfahren; 2008. https://doi.org/10.31030/1465413.

  37. 37.

    R Core Team. R: a language and environment for statistical computing. Vienna: R – Foundation for Statistical Computing; 2017. https://www.R-project.org/. Accessed 23 March 2019.

  38. 38.

    Eckmann R, Czerkies P, Helms C, Kleibs K. Evaluating the effectiveness of stocking vendace (Coregonus albula (L.)) eleutheroembryos by alizarin marking of otoliths. Adv Limnol. 1998;50:457–63.

  39. 39.

    Eckmann R. Alizarin marking of whitefish, Coregonus lavaretus otoliths during egg incubation. Fish Manag Ecol. 2003;10(4):233–9.

  40. 40.

    Baer J, Rösch R. Mass-marking of brown trout (Salmo trutta L.) larvae by alizarin: method and evaluation of stocking. J Appl Ichthyol. 2008;24:44–9.

  41. 41.

    Caudron A, Champigneulle A. Multiple marking of otoliths of brown trout, Salmo trutta L., with alizarin redS to compare efficiency of stocking of three early life stages. Fish Manag Ecol. 2009;16:219–24.

  42. 42.

    Lagardère F. Feasibility of otolith markings in large juvenile turbot, Scophthalmus maximus, using immersion in alizarin-red S solutions. ICES J Mar Sci. 2000;57(4):1175–81.

  43. 43.

    Liu Q, Zhang XM, Zhang PD, Nwafili SA. The use of alizarin red S and alizarin complexone for immersion marking Japanese flounder Paralichthys olivaceus (T.). Fish Res. 2009;98(1–3):67–74.

  44. 44.

    Beckman DW, Schulz RG. A simple method for marking fish otoliths with alizarin compounds. Trans Am Fish Soc. 1996;125:146–9.

  45. 45.

    Klein B. Versuchsplanung - DoE: Einführung in die Taguchi/Shainin-Methodik. 4th ed. De Gruyter Online; 2014.

  46. 46.

    Anonymus. Belastung von wildlebenden Flussfischen mit Dioxiden und PCB. Aktualisierte Stellungnahme* Nr. 027/2010 des BfR vom 16. Juni 2010. 2010. https://mobil.bfr.bund.de/.../belastung_von_wildlebenden_flussfischen_mit_dioxinen_und_pcb.pdf. Access 08 May 2019.

  47. 47.

    EFSA. Conclusion regarding the peer review of the pesticide risk assessment of the active substance dimethoate. EFSA Scientific Report. 2006;84:1–102 https://efsa.onlinelibrary.wiley.com/doi/pdf/10.2903/j.efsa.2006.84r. Accessed 16 May 2019.

  48. 48.

    EFSA. Anonymus. Coastal fisheries ordinance. 2017;§4. https://www.lav-mv.de/downloads/KueFVO_2017.pdf. Access 30 Apr 2019.

  49. 49.

    Åström M, Dekker W. When will the eel recover? A full life-cycle model. ICES J Mar Sci. 2007;64(1):1491–8.

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The authors wish to thank Dr. T. Schaarschmidt for establishing contact with the Department of Residue Analysis of the State Office for Agriculture, Food Safety and Fisheries.


This study was partly funded by the European Maritime Fisheries Fund (EMFF) and the Federal State Mecklenburg-Western Pomerania.

Author information

Correspondence to Laura Kullmann.

Ethics declarations

Prior to the study, the responsible Animal Protection Authority (State Office for Agriculture, Food Safety and Fisheries, Thierfelderstraße 18, 18059 Rostock, Germany) of the state Mecklenburg-Western Pomerania was contacted. Based on the consultation procedure, it was established that the present study complies with the Animal Welfare Regulation according to the national guidelines (Law on Animal Welfare §7a paragraph (2)3).

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The authors declare that they have no conflict of interest.

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Kullmann, L., Habedank, F., Kullmann, B. et al. Evaluation of the bioaccumulation potential of alizarin red S in fish muscle tissue using the European eel as a model. Anal Bioanal Chem 412, 1181–1192 (2020). https://doi.org/10.1007/s00216-019-02346-4

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  • Anguilla anguilla
  • Alizarin red S
  • LC-MS/MS
  • ARS concentration
  • Bioaccumulation potential
  • Fish marking