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Amphibians as sentinels of environmental pollution: from theory to practice Manuel Ortiz Santaliestra Instituto de Investigación en Recursos Cinegéticos.

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Presentation on theme: "Amphibians as sentinels of environmental pollution: from theory to practice Manuel Ortiz Santaliestra Instituto de Investigación en Recursos Cinegéticos."— Presentation transcript:

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

2 IUCN Red List of Threatened Species (2009) Major threats for amphibians

3 Evolution of ecotoxicology across Iberian herpetology meetings

4  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. State of the art of amphibian ecotoxicology

5 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…) SETAC Herps ecotoxicology advisory group (European section)

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

7 Ammonium nitrate effects on fish and amphibian aquatic stages No effects Sublethal effects Lethal effects 5030 10 7090 Index of N exposure [Log(conc*time)] Tolerant Sensitive  % 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 Tox 36:200; = (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 Thanks to Emilio López for the information and collection of field samples Adjuvants4 Growth regulators and stimulators7 Fungicides50 Herbicides23 Insecticides32 TOTAL116 3 OPs (chlorpyrifos, ethoprophos, methyl chlorpyrifos) 3 carbamates (fenoxicarb, indoxacarb, methiocarb) Agrochemicals approved for use on vineyards: 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

9 FISHAMPHIBIAN Major hematopoietic and lymphoid organs KidneyThymus Spleen Innate Immunity Humoral Skin (physical barrier)Skin (physical and chemical barrier) Mucous membranes (gut, gills)Mucous membranes (gut) Acute phase proteins (complement, c-reactive proteins, lectins) Acute phase proteins (complement, ??...) Cellular Macrophages Granulocytes (Eosinophils, Neutrophils) Non-specific citotoxic cellsNK cells Adaptive Immunity Humoral Antibodies (IgM, IgD, IgT)Antibodies (IgM, IgD, IgX, IgY, IgS) Cytokines (IL2, IL4, IL5, IL10, IL13, IFNγ) Cytokines (IL2, IL3, IL4, IL5, IL7, IFNγ) Cellular Th cell-type 1 & 2 responses Class I MHC positive-cellsClasses I and II MHC positive-cells 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 - Development of adult immune system components + Maternal antibodies Larval immune system Developing adult immune system Developed adult immune system Rearrangement of immune defenses Destruction of some components of the larval immune system Thyroid hormones Glucocorticoids Perchlorate Malathion* Atrazine* *Known immunotoxic effect during amphibian metamorphosis PCBs PCDDs 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 Adapted from Robert & Ohta (2009) Devel Dynam 238:1249. + - The problem of being born twice

11 Flame retardants Day 0 Dietary exposure to 0, 1, 6.1, 71.4 or 634 ng DE-71/g Larval development 50 Skin peptide assay Effects of PBDEs on innate immunity of juvenile R.pipiens Great Lakes Network Metamorphosis Juvenile 70 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 thioglycollate microbeads PBS Aspirate lavage fluid containing leukocytes 24 hours Lavage assays: technique Neutrophils Macrophages Coyle et al. (in prep.); Vatnick et al. (2006) Environ Toxicol Chem 25:199 Great Lakes Network

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

14 Skin peptide assay: technique Ortiz-Santaliestra & Karasov (in prep.); Rollins-Smith et al. (2002) Dev Comp Immunol 26:471; Sheafor et al. (2008) J Wildl Dis 44:226. Primary mechanism of defense against decline-related emergent diseases (i.e., chytridiomycosis) Negative control Peptide concentration Index of chytrid growth [Ln (OD 492nm ·10 2 )] Minimum inhibitory concentration (MIC) 95%CI 15 min (collecting buffer) Quantification (volume of pepttides / body mass) B. dendrobatidis growth inhibition test Norepinephrine (subcutaneous) Great Lakes Network

15 *** 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 Skin peptide assay: results Ortiz-Santaliestra & Karasov (in prep.) Great Lakes Network

16 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 Take-home message... *More than 1 ton is manufactured in or imported to the EU European Chemicals Agency,

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

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