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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
IUCN Red List of Threatened Species (2009) http://www.iucnredlist.org/initiatives/amphibians/analysis/major-threats
Evolution of ecotoxicology across Iberian herpetology meetings
Number
Percentage
17
6
4
7
2
2
Valencia
2000
Évora
2002
4
2
3
Málaga Donostia Coimbra
2004
2006
2008
0
Sevilla
2010
% of the total
8
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
X.laevis
R.pipiens
R.temporaria
Others
R.catesbeiana
R.clamitans
R.sylvatica
P.esculenta
A.maculatum
Schiesari et al. (2007) Conserv Biol 21:465; Grillitsch et al. (2009) Das Naturhist 3:10.
B.arenarum
B.americanus
B.bufo
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…)
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
Ammonium nitrate effects on fish and amphibian aquatic stages
No Sublethal
effects effects
Tolerant
Index of N exposure [Log(conc*time)]
Lethal effects
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 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.
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:
Adjuvants
Growth regulators and stimulators
Fungicides
Herbicides
Insecticides
TOTAL
4
7
50
23
32
116
Thanks to Emilio López for the information and collection of field samples
3 OPs (chlorpyrifos, ethoprophos, methyl chlorpyrifos)
3 carbamates (fenoxicarb, indoxacarb, methiocarb)
Why are not amphibians protected by fish toxicity assessment?
2) Problems associated to the immune system
FISH
Major hematopoietic and
lymphoid organs
Humoral
Innate
Immunity
Cellular
Adaptive
Immunity
Humoral
Cellular
AMPHIBIAN
Kidney
Thymus
Spleen
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, ??...)
Macrophages
Macrophages
Granulocytes (Eosinophils,
Neutrophils)
Granulocytes (Eosinophils,
Neutrophils)
Non-specific citotoxic cells
NK cells
Antibodies (IgM, IgD, IgT)
Antibodies (IgM, IgD, IgX, IgY, IgS)
Cytokines (IL2, IL4, IL5, IL10,
IL13, IFNγ)
Cytokines (IL2, IL3, IL4, IL5, IL7,
IFNγ)
Th cell-type 1 & 2 responses
Th cell-type 1 & 2 responses
Class I MHC positive-cells
Classes I and II MHC positive-cells
Rubio-Godoy (2010) Rev Mex Cienc Pec 1:47; Robert & Ohta (2009) Devel Dynam 238:1249.
The problem of being born twice
Adapted from Robert & Ohta (2009) Devel Dynam 238:1249.
Maternal
antibodies
Larval immune
system
Developing adult
immune system
Developed adult
immune system
Rearrangement of immune defenses
Development of adult
immune system components
+
Destruction of some components
+
of the larval immune system
Thyroid hormones
-
Perchlorate
Malathion*
Atrazine*
Glucocorticoids
-
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
*Known
immunotoxic
effect during
amphibian
metamorphosis
Effects of PBDEs on innate immunity of juvenile R.pipiens
Great Lakes Network
Larval development
Metamorphosis
Juvenile
Flame retardants
Day 0
Dietary exposure to
0, 1, 6.1, 71.4 or 634
ng DE-71/g
Coyle et al. (in prep.); Ortiz-Santaliestra & Karasov (in prep.)
50
Skin peptide assay
70
Lavage assays
Neutrophil recruitment
Phagocitosis
Lavage assays: technique
Great Lakes Network
1) Neutrophil recruitment: characterization of inflammatory response after
i.p. injection of thioglycollate
2) Measurement of phagocytic activity using 1 µm FITC-labeled microbeads
PBS
•thioglycollate
•microbeads
24 hours
Neutrophils
Coyle et al. (in prep.); Vatnick et al. (2006) Environ Toxicol Chem 25:199
Aspirate lavage
fluid containing
leukocytes
Macrophages
Lavage assays: results
Great Lakes Network
p = 0.0759
p = 0.2151
None
Low
Medium
Highly
Percent Phagocytosis
100
Coyle et al. (in prep.)
80
60
40
20
0
CTL
1
6.1
71.4
634
Larval treatment (DE-71 ng/g)
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)
Index of chytrid growth
[Ln (OD492nm·102)]
2,2
B. dendrobatidis growth
inhibition test
Minimum inhibitory
concentration (MIC)
1,9
1,6
1,3
95%CI
1
1
10
100
Peptide concentration
1000
Ortiz-Santaliestra & Karasov (in prep.); Rollins-Smith et al. (2002) Dev Comp Immunol 26:471; Sheafor et al. (2008)
J Wildl Dis 44:226.
Negative 10000
control
Skin peptide assay: results
[peptides] +-SE
140
120
100
80
***
NS
Great Lakes Network
0 ng/g
1 ng/g
6.1 ng/g
60
40
71.4 ng/g
634 ng/g
20
0
Extracted / mass
MIC*0.01
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.)
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/
Collaborators
Funding sources
Emilio López
Tawnya Coyle (UW Madison)
Jeff Lorch (UW Madison)
Bill Karasov (UW Madison)
THANK YOU
Great Lakes Network