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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 [email protected]. Major threats for amphibians. - PowerPoint PPT Presentation
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Amphibians as sentinels of environmental pollution: from theory to practice
Manuel Ortiz Santaliestra
Instituto de Investigación en Recursos Cinegéticos UCLM-CSIC-JCCMCiudad Real, Spain
IUCN Red List of Threatened Species (2009) http://www.iucnredlist.org/initiatives/amphibians/analysis/major-threats
Major threats for amphibians
Evolution of ecotoxicology across Iberian herpetology meetings
7
3
17
422
Valencia2000
Évora2002
Málaga2004
Donostia2006
Coimbra2008
Sevilla2010
0
2
4
6
8
% o
f th
e t
ota
l
Number Percentage
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.
X.laevis
R.pipiens
Others
R.catesbeiana
R.temporaria
B.arenarum
R.sylvatica
B.americanus
B.bufoA.maculatum
P.esculenta
R.clamitans
EurasiaN AmericaS America
Schiesari et al. (2007) Conserv Biol 21:465; Grillitsch et al. (2009) Das Naturhist 3:10.
State of the art of amphibian ecotoxicology
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)
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
Ammonium nitrate effects on fish and amphibian aquatic stages
No effects
Sublethal effects
Lethal effects
503010 70 90
Ind
ex o
f N
exp
osu
re [
Lo
g(c
on
c*ti
me)
]
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.
Thanks to Emilio López for the information and collection of field samples
Adjuvants 4Growth regulators and stimulators 7Fungicides 50Herbicides 23Insecticides 32
TOTAL11
6
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
FISH AMPHIBIAN
Major hematopoietic and lymphoid organs
Kidney ThymusSpleen
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 Macrophages
Granulocytes (Eosinophils, Neutrophils)
Granulocytes (Eosinophils, Neutrophils)
Non-specific citotoxic cells NK 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
Th cell-type 1 & 2 responses
Class I MHC positive-cells Classes 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.
-Development of adult immune system
components
+
Maternal antibodies
Larval immune system
Developing adult immune system
Developed adult immune
systemRearrangement of immune defenses
Destruction of some components of the larval
immune system
Thyroid hormones
Glucocorticoids
PerchlorateMalathion*Atrazine*
*Known immunotoxic effect during
amphibian metamorphosis
PCBsPCDDs
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
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.pipiensGreat Lakes Network
Metamorphosis Juvenile
70
Lavage assaysNeutrophil recruitmentPhagocitosis
Coyle et al. (in prep.); Ortiz-Santaliestra & Karasov (in prep.)
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
Lavage assays: results
p = 0.0759
p = 0.2151
Coyle et al. (in prep.)
Great Lakes Network
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)
1
1,3
1,6
1,9
2,2
1 10 100 1000 10000 Negative control
Peptide concentration
Inde
x of
chy
trid
gro
wth
[L
n (O
D49
2nm·1
02 )]
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
0
20
40
60
80
100
120
140
Extracted / mass MIC*0.01
[pep
tid
es]
+-S
E 0 ng/g
1 ng/g
6.1 ng/g
71.4 ng/g
634 ng/g
*** 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
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, http://echa.europa.eu/
Great Lakes Network
Emilio López
Tawnya Coyle (UW Madison)
Jeff Lorch (UW Madison)
Bill Karasov (UW Madison)
Collaborators Funding sources
THANK YOU