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Amphibians as sentinels of environmental pollution: from theory to practice

Amphibians as sentinels of environmental pollution: from theory to practice. Manuel Ortiz Santaliestra Instituto de Investigación en Recursos Cinegéticos UCLM-CSIC-JCCM Ciudad Real, Spain ManuelE.Ortiz@uclm.es. Major threats for amphibians.

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Amphibians as sentinels of environmental pollution: from theory to practice

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  1. Amphibians as sentinels of environmental pollution: from theory to practice Manuel Ortiz Santaliestra Instituto de Investigación en Recursos Cinegéticos UCLM-CSIC-JCCM Ciudad Real, Spain ManuelE.Ortiz@uclm.es

  2. Major threats for amphibians IUCN Red List of Threatened Species (2009) http://www.iucnredlist.org/initiatives/amphibians/analysis/major-threats

  3. Evolution of ecotoxicology across Iberian herpetology meetings

  4. State of the art of amphibian ecotoxicology • The eleven most widely distributed amphibian species account for > 50 % of all ecotoxicological studies. • Of those species suggested to be threatened by pollution in the GAA (N=1100), only 68 (6.2 %) were subject to any ecotoxicological study. Eurasia N America S America Schiesari et al. (2007) Conserv Biol 21:465; Grillitsch et al. (2009) Das Naturhist 3:10.

  5. SETAC Herps ecotoxicology advisory group (European section) RESEARCH NEEDS Use experimental designs to maximize the ecological relevance of results (e.g., mesocosms, field studies, native species or populations…) Stimulate research about impacts of pollution on areas of high herpetological biodiversity and endangered species or populations Develop tools for ecotoxicological assessment suitable for herps (e.g., specific biomarkers. GIS…) MANAGEMENT NEEDS Consider amphibian & reptile toxicity data for establishing quality criteria and approving the use of chemical substances Consider herp biological features in the timing of chemical release to the environment (e.g., avoid application of agrochemicals during the amphibian early larval stages or during the reptile egg incubation) Protect herp habitats from pollution also at a microecological scale (e.g., breeding ponds for amphibians…)

  6. Widlife toxicity data used for registration of chemical substances TERRESTRIAL AQUATIC Are fish good surrogates to estimate sensitivity of amphibians to pollutants? Because their naked skins, amphibians would be more sensitive than fishes to pollution

  7. Ammonium nitrate effects on fish and amphibian aquatic stages Sublethal effects No effects Lethal effects Tolerant Index of N exposure [Log(conc*time)] Sensitive 10 30 50 70 90  % mortality Berger (1989) Ecol Int Bull 17:65; Capkin et al (2010) Tur J Fish Aq Sci 10:19; Hamer et al (2004) Agr Ecos Env 102:299; Hecnar (1995) Env Tox Chem 14:2131; Ortiz et al (2004) Arch Env Contam Tox 47:234; Ortiz-Santaliestra et al (2006) Env Tox Chem 25:105; = (2007) Aq Tox 85:251; = (2010) Env Poll 158:934; =(2010) Aq Tox 99:198; = (in press) Arch Env Contam Tox; Puglis & Boone (2007) Env Tox Chem 26:2198; Schuytema & Nebeker (1999)Arch Env Contam Tox36:200; =(1999) Env Tox Chem 18:2251; Watt & Jarvis (1997) Ecotox 6:55; Watt & Oldham (1995) Freshw Biol 33:319; Xu & Oldham (1997) Arch Env Contam Tox 32:298.

  8. Why are not amphibians protected by fish toxicity assessment? 1) Problems associated to temporary ponds P. waltl mass mortality (August 2010) San Carlos del Valle, Ciudad Real Agrochemicals approved for use on vineyards: 3 OPs(chlorpyrifos, ethoprophos, methyl chlorpyrifos) 3 carbamates(fenoxicarb, indoxacarb, methiocarb) Thanks to Emilio López for the information and collection of field samples

  9. Why are not amphibians protected by fish toxicity assessment? 2) Problems associated to the immune system Rubio-Godoy (2010) Rev Mex Cienc Pec 1:47; Robert & Ohta (2009) Devel Dynam 238:1249.

  10. The problem of being born twice Adapted from Robert & Ohta (2009) Devel Dynam 238:1249. Developing adult immune system Maternal antibodies Larval immune system Developed adult immune system Rearrangement of immune defenses Perchlorate Malathion* Atrazine* Development of adult immune system components *Known immunotoxic effect during amphibian metamorphosis + - Thyroid hormones Destruction of some components of the larval immune system PCBs PCDDs + Glucocorticoids - Cheek (2006) Rev Biol Trop 54(s1):1; Fordham et al. (2001) Vet Immunol Immunopath 20:179; Kiesecker (2002) PNAS 99:9900; Ortiz-Santaliestra & Sparling (2007) Archiv Environ Comtam Toxicol 53:639; Rollins-Smith et al. (1997) Dev Immunol 5:145. Sullivan & Spence (2003) Vet Immunol Immunopath 22:627

  11. Effects of PBDEs on innate immunity of juvenile R.pipiens Great Lakes Network Juvenile Metamorphosis Larval development Flame retardants 50 70 Day 0 Dietary exposure to 0, 1, 6.1, 71.4 or 634 ng DE-71/g Skin peptide assay Lavage assays Neutrophil recruitment Phagocitosis Coyle et al. (in prep.); Ortiz-Santaliestra & Karasov (in prep.)

  12. 1) Neutrophil recruitment: characterization of inflammatory response after i.p. injection of thioglycollate 2) Measurement of phagocytic activity using 1 µm FITC-labeled microbeads Lavage assays: technique Great Lakes Network PBS • thioglycollate • microbeads Aspirate lavage fluid containing leukocytes 24 hours Neutrophils Macrophages Coyle et al. (in prep.); Vatnick et al. (2006) Environ Toxicol Chem 25:199

  13. Lavage assays: results Great Lakes Network p = 0.0759 p = 0.2151 Coyle et al. (in prep.)

  14. Minimum inhibitory concentration (MIC) Skin peptide assay: technique Great Lakes Network Primary mechanism of defense against decline-related emergent diseases (i.e., chytridiomycosis) Quantification (volume of pepttides / body mass) Norepinephrine (subcutaneous) 15 min (collecting buffer) B. dendrobatidis growth inhibition test Index of chytrid growth [Ln (OD492nm·102)] 95%CI Negative control Peptide concentration Ortiz-Santaliestra & Karasov (in prep.); Rollins-Smith et al. (2002) Dev Comp Immunol 26:471; Sheafor et al. (2008) J Wildl Dis 44:226.

  15. Skin peptide assay: results Great Lakes Network *** NS • Out of the three immuncompetence indicators tested, the only affected by PBDE exposure was… • the only that can not be tested in fish • the only that has been directly involved in disease-mediated declines Ortiz-Santaliestra & Karasov (in prep.)

  16. Take-home message... Herpetologists interested in ecotoxicology may ask to or become part of the advisory group There is too much left to do, and protecting amphibians from pollution requires herpetologists’ work 143,000 chemical substances have been pre-registered by REACH*. Protecting amphibians from the potential risks posed by these substances is a key question to slow down declines Advancing in reptile ecotoxicology and assessing the situation of reptiles with regards to environmental pollution must be an imminent step *More than 1 ton is manufactured in or imported to the EU European Chemicals Agency, http://echa.europa.eu/

  17. Collaborators Funding sources Emilio López Tawnya Coyle (UW Madison) Jeff Lorch (UW Madison) Bill Karasov (UW Madison) Great Lakes Network THANK YOU

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