PHAGOCYTIC ACTIVITY OF EARTHWORM PHAGOCYTIC ACTIVITY OF EARTHWORM COELOMYCYTES USED AS ACOELOMYCYTES USED AS A

SUBLETHAL BIOMARKER OF METAL SUBLETHAL BIOMARKER OF METAL TOXICITYTOXICITY

Sauvé S, Hendawi M, Brousseau P, Fournier MSauvé S, Hendawi M, Brousseau P, Fournier M

Département de chimie – Université de MontréalINRS-Institut Armand Frappier-Santé Humaine

© Sauvé 2001

EarthwormsEarthworms

Earthworms lead an essential ecological role for maintaining soil health, fertility, structure, nutrient cycling, …

Standard worm assays evaluate lethality

Sublethal markers such as reproduction and immunocompetence can be quantified

© Sauvé 2001

Worm speciesWorm species

Terrestrial earthworms

Lumbricus terrestris

Eisenia fetida

Aporrectodea turgida

Aquatic worm

Tubifex tubifex

© Sauvé 2001

Cell ExtrusionCell Extrusion

By puncture with pasteur pipette or syringe

Ethanol extrusion

Electrical stimulation

Ultrasound

Extruded cells are suspended in a Lumbricus Basal Salt Solution (LBSS)

© Sauvé 2001

Species-specific parametersSpecies-specific parameters

 

E. fetida L. terrestris A. turgida T. tubifex

Fresh weight

(g)

0.37 4.24 0.54 0.005

Total cells /

animals (106)

2.65 13.4 3.28 0.14

Viability (%)

Trypan Blue

81.9 87.4 77.7 76.2

Viability (%)

PI

66.3 74.8 90.9 74.1

© Sauvé 2001

PhagocytosisPhagocytosis

Earthworm coelomocytes (Eisenia fetida)

© Sauvé 2001

Flow CytometryFlow Cytometry

100 101 102 103 104

FL1-Height

0128

Events

Gm: 363.33 CV: 23.12 [72-255] 8601 (86.0 %)

M1

100 101 102 103 104

FL1-Height

0256

Events

Gm: 43.08 CV: 26.20 [72-255] 2544 (25.4 %)

M1

0 1023

SS

C-H

eigh

t0

1023

FSC-Height

© Sauvé 2001

PhagocytosisPhagocytosis

0 255

SS

C-H

eig

ht

10

010

110

210

310

4

FSC-Height

R1

0 255

SS

C-H

eig

ht

10

010

110

210

310

4

FSC-Height

R1

0 255

SS

C-H

eig

ht

10

010

110

210

310

4

FSC-Height

R1

0 255

SS

C-H

eig

ht

10

010

110

210

310

4

FSC-Height

R1

Species Control Toxic (10-4M HgCl2)

L. terrestris

E. fetida

© Sauvé 2001

Silver and ArsenicSilver and Arsenic

-10 -9 -8 -7 -6 -5 -4 -30

255075

100125150175

L. terrestrisE. fetida

-10 -9 -8 -7 -6 -5 -4 -3

AgNO3 (10x M)

0255075

100125150175

Ph

ag

ocy

tosi

s ( %

)

-10 -9 -8 -7 -6 -5 -4 -30

25

50

75

100

125

-10 -9 -8 -7 -6 -5 -4 -3

AgNO3 (10x M)

0

25

50

75

100

125

Via

bili

t y (

%)

-10 -9 -8 -7 -6 -5 -4 -30

255075

100125150175

-10 -9 -8 -7 -6 -5 -4 -3

Na2HAsO4 (10x M)

0255075

100125150175

Ph

ag

ocy

tosi

s ( %

)

-10 -9 -8 -7 -6 -5 -4 -30

25

50

75

100

125

-10 -9 -8 -7 -6 -5 -4 -3

Na2HAsO4 (10x M)

0

25

50

75

100

125

Via

bili

t y (

%)

© Sauvé 2001

Cadmium and CopperCadmium and Copper

-10 -9 -8 -7 -6 -5 -4 -30

255075

100125150175

-10 -9 -8 -7 -6 -5 -4 -3

CdCl2 (10x M)

0255075

100125150175

Ph

ag

ocy

tosi

s ( %

)

-10 -9 -8 -7 -6 -5 -4 -30

25

50

75

100

125

-10 -9 -8 -7 -6 -5 -4 -3

CdCl2 (10x M)

0

25

50

75

100

125

Via

bili

ty (

%)

-10 -9 -8 -7 -6 -5 -4 -30

255075

100125150175

T. tubifexA. turgidaL. terrestrisE. fetida

-10 -9 -8 -7 -6 -5 -4 -3

CuCl2 (10x M)

0255075

100125150175

Ph

ag

ocy

tosi

s ( %

)

-10 -9 -8 -7 -6 -5 -4 -30

25

50

75

100

125

-10 -9 -8 -7 -6 -5 -4 -3

CuCl2 (10x M)

0

25

50

75

100

125

Via

bili

ty (

%)

© Sauvé 2001

MercuryMercury

-10 -9 -8 -7 -6 -5 -4 -30

255075

100125150175

-10 -9 -8 -7 -6 -5 -4 -3

HgCl2 (10x M)

0255075

100125150175

Ph

ag

ocy

tosi

s ( %

)

-10 -9 -8 -7 -6 -5 -4 -30

25

50

75

100

125

-10 -9 -8 -7 -6 -5 -4 -3

HgCl2 (10x M)

0

25

50

75

100

125

Via

bili

ty (

%)

-10 -9 -8 -7 -6 -5 -4 -30

255075

100125150175

T. tubifexA. turgidaL. terrestrisE. fetida

-10 -9 -8 -7 -6 -5 -4 -3

CH3HgCl (10x M)

0255075

100125150175

Ph

ag

ocy

tosi

s ( %

)

-10 -9 -8 -7 -6 -5 -4 -30

25

50

75

100

125

-10 -9 -8 -7 -6 -5 -4 -3

CH3HgCl (10x M)

0

25

50

75

100

125

Via

bili

ty (

%)

© Sauvé 2001

Lead and ZincLead and Zinc

-10 -9 -8 -7 -6 -5 -4 -30

255075

100125150175

-10 -9 -8 -7 -6 -5 -4 -3

PbCl2 (10x M)

0255075

100125150175

Ph

ag

ocy

tosi

s ( %

)

-10 -9 -8 -7 -6 -5 -4 -30

25

50

75

100

125

-10 -9 -8 -7 -6 -5 -4 -3

PbCl2 (10x M)

0

25

50

75

100

125

Via

bili

ty (

%)

-10 -9 -8 -7 -6 -5 -4 -30

255075

100125150175

A. turgidaL. terrestrisE. fetida

-10 -9 -8 -7 -6 -5 -4 -3

ZnCl2 (10x M)

0255075

100125150175

Ph

ag

ocy

tosi

s ( %

)

-10 -9 -8 -7 -6 -5 -4 -30

25

50

75

100

125

-10 -9 -8 -7 -6 -5 -4 -3

ZnCl2 (10x M)

0

25

50

75

100

125V

iab

ility

(%

)

© Sauvé 2001

In vitroIn vitro Toxicity thresholds Toxicity thresholdsCompound EC50

(mg L-1)

AgNO3 6.6

Na2HAsO4 ND

CdCl2 2.4

CoCl2 ND

KCr2O7 ND

CuCl2 1.5

HgCl2 1.0

Compound EC50

(mg L-1)

CH3HgCl 0.4

MoCl4 47.5*

NiCl2 6.4

PbCl2 134*

NaSeO3 51447*

ZnCl2 24853*

ND = not detected * = extrapolated

© Sauvé 2001

Transfer Transfer in vitroin vitro to to in vivoin vivo

In vitro cellular exposure

Petri dish, solution exposure

Soil incubation (using the OECD reproduction protocol)

Field, in situ exposure in mesocosm « worm bags »

Some recent field applications to the evaluation of cement factory emissions