Amphibians as sentinels of environmental pollution: from theory to practice

Manuel Ortiz Santaliestra

Instituto de Investigación en Recursos Cinegéticos UCLM-CSIC-JCCMCiudad Real, Spain

ManuelE.Ortiz@uclm.es

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