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The Effect of Esfenvalerate and Prochloraz on Amphibians with special reference to
Xenopus laevis and Bombina bombina
Appendices 1-4
Appendix 1
Score sheet of malformations after 96 h
Click here to see score sheet
Appendix 2
The composition of the culture media
FETAX solution water
| 625 mg |
NaCl |
| 96 mg |
NaHCO3 |
| 30 mg |
Kcl |
| 15 mg |
CaCl2 |
| 60 mg |
CaSO42H20 |
| 75 mg |
MgSO4 |
per litre of deionised or distilled water. The pH of the final solution should be 7.6 to 7.9. All chemicals were reagent-grade or better.
Appendix 3
Comparison of the LC50 values from the present amphibian test with the results from other tests with amphibians and the standard aquatic test organisms.
Table A3.1
The effects of the test chemical esfenvalerate.
| Organisms |
Endpoint |
Time
(h) |
Concentration
(mg/l) |
Note |
Ref. |
Fathead
Minnows |
LC50 |
96 |
0.0007 |
|
Pesticide
manual |
| Bluegill |
LC50 |
96 |
0.0002 |
flowing
system |
Danish
EPA |
| Killifish |
LC50 |
96 |
0.002 |
postlarval
stage |
Danish
EPA |
| Killifish |
LC50 |
96 |
0.002 |
adult
stage |
Danish
EPA |
| |
| Daphnia |
EC50 |
48 |
0.00024 |
|
Pesticide
manual |
| |
Rana
Pipiens |
LC50 |
22 |
> 0.1 |
tadpoles |
Berrill
et al. 1993 |
Rana
Clamitans |
LC50 |
22 |
> 0.1 |
tadpoles |
Berrill
et al. 1993 |
Leopard frog
(Rana spp) |
LC50 |
96 |
0.007 |
tadpoles |
Materna et
al. 1995 |
| |
Xenopus
Laevis |
LC50 |
96 |
>0.15 |
embryos |
present study |
Bombina
Bombina |
LC50 |
96 |
>0.15 |
embryos |
present
study |
Table A3.2
The effects of the test chemical prochloraz.
| Organisms |
Endpoint |
Time |
Concentration
(mg/l) |
Note |
Ref. |
Rainbow
trout |
LC50 |
96 h |
1.0 |
|
Pesticide
manual |
| Bluegill |
LC50 |
96 h |
2.2 |
|
Pesticide
manual |
Salmo
gairdneri |
LC50 |
96 h |
1.5 |
flow-
through |
Danish
EPA |
Bluegill
sunfish |
LC50 |
96 h |
2.2 |
static
test |
Danish
EPA |
Harlequin
fish |
LC50 |
96 h |
2.8 |
flow-
through |
Danish
EPA |
| |
Daphnia
magna |
LC50 |
48 h |
2.6 |
|
Danish
EPA |
Daphnia
magna |
LC50 |
21 days |
0.047 |
chronic
test |
Danish
EPA |
| |
| Scenedesmus |
EC50 |
96 h |
0.073 |
|
Danish
EPA |
| |
| Xenopus laevis |
LC50 |
96 h |
4.5 |
|
present
study |
Bombina
bombina |
LC50 |
96 h |
> 10 |
|
present
study |
Appendix 4
Table A4.1
The effect of esfenvalerate in Xenopus laevis on malformation, growth inhibition and mortality. Mean of 3 experiments.
Effeketen af esfenvalerat på Xenopus laevis illustreret ved sublethale defekter, væksthæmning og dødelighed. Middeltal af 3 forsøg.
| Concentrations |
Malformation
( in procent) |
Growth inhibition
(in m.m.) |
Mortality
(in procent) |
| Control |
3.2 (S.D. = 1.3) |
10.1 (S.D. = 0.4) |
1.7 (S.D. = 2.4) |
| DMSO control |
2.9 (S.D. = 1.5) |
9.9 (S.D. = 0.2) |
0.0 (S.D. = 0.0) |
| 1.0 µg/l |
3.3 (S.D. = 3.4) |
9.9 (S.D. = 0.2) |
1.3 (S.D. = 0.9) |
| 2.5 µg/l |
5.1 (S.D. = 0.7) |
9.8 (S.D. = 0.5) |
1.3 (S.D. = 1.9) |
| 5.0 µg/l |
88.0 (S.D. = 16.9) |
9.4 (S.D. = 0.5) |
0.0 (S.D. = 0.0) |
| 10.0 µg/l |
100.0 (S.D. = 0.0) |
8.4 (S.D. = 0.1) |
0.7 (S.D. = 0.9) |
| 50.0 µg/l |
100.0 (S.D. = 0.0) |
8.3 (S.D. = 0,2) |
1.3 (S.D. = 0.9) |
| 100 µg/l |
100.0 (S.D. = 0.0) |
8.6 (S.D. = 0.2) |
15.7 (S.D. = 4.9) |
| 150 µg/l |
100.0 (S.D. = 0.0) |
8.5 (S.D. = 0.3) |
16.0 (S.D. = 8.6) |
Table A4.2
The effect of esfenvalerate in Xenopus laevis on malformation, growth inhibition and mortality. The results are expressed as NOEC, LOEC and EC50/LC50 after 96 h of exposure.
Effekten af esfenvalerat på Xenopus laevis illustreret ved sublethale defekter, væksthæmning og dødelighed. Resultaterne er udtrykt ved NOEC, LOEC, EC50/LC50 efter 96 timers eksponering.
| |
Malformations |
| NOEC |
LOEC |
EC50 |
| Means of 3 experiments |
2.0 µg/l |
5.0 µg/l |
3.0 µg/l |
| Standard deviations |
0.7 |
0.0 |
1.0 |
| The 95% confidence limits |
1.2 -2.8 |
- |
1.9-4.1 |
| |
Growth inhibition |
| NOEC |
LOEC |
EC50 |
| Means of 3 experiments |
5 µg/l |
10 µg/l |
150 µg/l> |
| Standard deviations |
0.0 |
0.0 |
0.0 |
| The 95% confidence limits |
- |
- |
- |
| |
Mortality |
| NOEC |
LOEC |
LC50 |
| Means of 3 experiments |
50 µg/l |
100 µg/l |
150 µg/l> |
| Standard deviations |
0.0 |
0.0 |
0.0 |
| The 95% confidence limits |
- |
- |
- |
Table A4.3
The effect of prochloraz in Xenopus laevis on malformation, growth inhibition and mortality in experiments where the jelly coat not has been removed. Mean of 3 experiments.
Effekten af procloraz i Xenopus laevis med sublethale defekter, væksthæmning og dødelighed i eksperimenter, hvor gelékappen ikke er blevet fjernet. Middeltal af 3 forsøg.
| Concentrations |
Malformation
( in procent) |
Growth inhibition
(in m.m.) |
Mortality
(in procent) |
| Control |
2.3 (S.D. = 1.8) |
9.6 (S.D. = 0.2) |
1.7 (S.D. = 1.7) |
| Acetone control |
3.0 (S.D. = 0.8) |
9.7 (S.D. = 0.4) |
1.7 (S.D. = 1.2) |
| 0.1 mg/l |
3.0 (S.D. = 0.8) |
9.4 (S.D. = 0.2) |
2.3 (S.D. = 0.5) |
| 1.0 mg/l |
12.0 (S.D. = 5.1) |
9.2 (S.D. = 0.2) |
0.0 (S.D. = 0.0) |
| 1.5 mg/l |
60.7 (S.D. = 14.4) |
8.9 (S.D. = 0.6) |
2.3 (S.D. = 1.7) |
| 2.0 mg/l |
100.0 (S.D. = 0.0) |
8.3 (S.D. = 0.3) |
2.3 (S.D. = 2.1) |
| 3.0 mg/l |
100.0 (S.D. = 0.0) |
7.5 (S.D. = 0.3) |
2.0 (S.D. = 2.8) |
| 5.0 mg/l |
100.0 (S.D. = 0.0) |
7.6 (S.D. = 0.5) |
60.7 (S.D. = 31.3) |
| 10 mg/l |
- |
- |
100.0 (S.D. = 0.0) |
Table A4.4
The effect of prochloraz in Xenopus laevis on malformation, growth inhibition and mortality in experiments where the jelly coat not has been removed. The results are expressed as NOEC, LOEC and EC50/LC50 after 96 h of exposure.
Effekten af prochloraz på Xenopus laevis illustreret ved sublethale defekter, væksthæmning og dødelighed i eksperimenter, hvor gelékappen ikke er blevet fjernet. Resultaterne er udtrykt ved NOEC, LOEC, EC50/LC50 efter 96 timers eksponering.
| |
Malformations |
| NOEC |
LOEC |
EC50 |
| Means of 3 experiments |
0.4 mg/l |
1.2 mg/l |
1.3 mg/l |
| Standard deviations |
0.4 |
0.2 |
0.05 |
| The 95% confidence limits |
0.0-0.9 |
0.9-1.5 |
1.25-1.35 |
| |
Growth inhibition |
| NOEC |
LOEC |
EC50 |
| Means of 3 experiments |
1.8 mg/l |
2.3 mg/l |
5 mg/l> |
| Standard deviations |
0.2 |
0.5 |
0.0 |
| The 95% confidence limits |
1.5-2.1 |
1.8-2.8 |
- |
| |
Mortality |
| NOEC |
LOEC |
LC50 |
| Means of 3 experiments |
3 mg/l |
5 mg/l |
4.6 mg/l |
| Standard deviations |
0.0 |
0.0 |
0.9 |
| The 95% confidence limits |
- |
- |
3.6-5.6 |
Table A4.5
The effect of prochloraz in Xenopus laevis on malformation, growth inhibition and mortality in standard experiments where the jelly coat has been removed. Mean of 3 experiments.
Effekten af procloraz på Xenopus laevis illustreret ved sublethale defekter, væksthæmning og dødelighed i eksperimenter, hvor gelékappen er blevet fjernet. Middeltal af 3 forsøg.
| Concentrations |
Malformation
( in procent) |
Growth inhibition
(in m.m.) |
Mortality
(in procent) |
| Control |
2.3 (S.D. = 1.9) |
9.0 (S.D. = 0.3) |
0.3 (S.D. = 0.5) |
| Acetone control |
4.7 (S.D. = 2.1) |
9.4 (S.D. = 0.2) |
1.0 (S.D. = 1.4) |
| 0.1 mg/l |
1.3 (S.D. = 1.9) |
9.6 (S.D. = 0.1) |
0.7 (S.D. = 0.9) |
| 1.0 mg/l |
10.7 (S.D. = 2.1) |
9.1 (S.D. = 0.1) |
0.0 (S.D. = 0.0) |
| 1.5 mg/l |
57.3 (S.D. = 12.3) |
8.9 (S.D. = 0.3) |
0.0 (S.D. = 0.0) |
| 2.0 mg/l |
100.0 (S.D. = 0.0) |
8.8 (S.D. = 0.4) |
0.7 (S.D. = 0.9) |
| 3.0 mg/l |
100.0 (S.D. = 0.0) |
7.7 (S.D. = 0.3) |
4.7 (S.D. = 4.1) |
| 5.0 mg/l |
100.0 (S.D. = 0.0) |
7.6 (S.D. = 0.4) |
61.7 (S.D. = 29.3) |
| 10 mg/l |
- |
- |
100.0 (S.D. = 0.0) |
Table A4.6
The effect of prochloraz in Xenopus laevis on malformation, growth inhibition and mortality in standard experiments where the jelly coat has been removed. The results are expressed as NOEC, LOEC and EC50/LC50 after 96 h of exposure.
Effekten af prochloraz på Xenopus laevis illustreret ved sublethale defekter, væksthæmning og dødelighed i eksperimenter, hvor gelékappen ikke er blevet fjernet. Resultaterne er udtrykt ved NOEC, LOEC, EC50/LC50 efter 96 timers eksponering.
| |
Malformations |
| NOEC |
LOEC |
EC50 |
| Means of 3 experiments |
0.1 mg/l |
1.0 mg/l |
1.4 mg/l |
| Standard deviations |
0.0 |
0.0 |
0.1 |
| The 95% confidence limits |
- |
- |
1.3 - 1.5 |
| |
Growth inhibition |
| NOEC |
LOEC |
EC50 |
| Means of 3 experiments |
2 mg/l |
3 mg/l |
5 mg/l > |
| Standard deviations |
0.0 |
0.0 |
0.0 |
| The 95% confidence limits |
- |
- |
- |
| |
Mortality |
| NOEC |
LOEC |
LC50 |
| Means of 3 experiments |
3 mg/l |
5 mg/l |
4.5 mg/l |
| Standard deviations |
0.0 |
0.0 |
0.6 |
| The 95% confidence limits |
- |
- |
3.9- 5.1 |
Table A4.7
The effect of prochloraz in Bombina bombina on malformation, growth inhibition and mortality. Mean of 3 experiments.
Effekten af procloraz på Bombina bombina illustreret ved sublethale defekter, væksthæmning og dødelighed. Middeltal af 3 forsøg.
| Concentrations |
Malformation
( in procent) |
Growth inhibition
(in m.m.) |
Mortality
(in procent) |
| Control |
5.0 (S.D. = 4.1) |
9.1 (S.D. = 0.2) |
0.8 (S.D. = 1.2) |
| Acetone control |
3.0 (S.D. = 2.1) |
9.2 (S.D. = 0.2) |
0.0 (S.D. = 0.0) |
| 1.0 mg/l |
6.2 (S.D. = 8.4) |
8.8 (S.D. = 0.4) |
0.0 (S.D. = 0.0) |
| 1.5 mg/l |
22.0 (S.D. = 7.0) |
8.7 (S.D. = 0.3) |
0.8 (S.D. = 1.2) |
| 2.0 mg/l |
42.3 (S.D. = 8.8) |
8.5 (S.D. = 0.3) |
3.2 (S.D. = 2.2) |
| 5.0 mg/l |
100.0 (S.D. = 0.0) |
7.4 (S.D. = 0.4) |
8.3 (S.D. = 6.2) |
| 10 mg/l |
100.0 (S.D. = 0.0) |
6.5 (S.D. = 0.2) |
28.0 (S.D. = 22.0) |
Table A4.8
The effect of prochloraz in Bombina bombina on malformation, growth inhibition and mortality. The results are expressed as NOEC, LOEC and EC50/LC50 after 120 h of exposure.
Effekten af prochloraz i Bombina bombina udtrykt ved sublethale defekter, væksthæmning og dødelighed. Resultaterne er udtrykt som NOEC, LOEC og EC50/LC50 efter 120 timers eksponering.
| |
Malformations |
| NOEC |
LOEC |
EC50 |
| Means of 3 experiments |
1 mg/l |
1.5 mg/l |
2.1 mg/l |
| Standard deviations |
0.0 |
0.0 |
0.1 |
| The 95% confidence limits |
- |
- |
1.9 - 2.3 |
| |
Growth inhibition |
| NOEC |
LOEC |
EC50 |
| Means of 3 experiments |
2 mg/l |
5 mg/l |
10 mg/l> |
| Standard deviations |
0.0 |
0.0 |
0.0 |
| The 95% confidence limits |
- |
- |
- |
| |
Mortality |
| NOEC |
LOEC |
LC50 |
| Means of 3 experiments |
2.5mg/l |
5 mg/l |
10 mg/l> |
| Standard deviations |
0.0 |
0.0 |
0.0 |
| The 95% confidence limits |
- |
- |
- |
Table A4.9
The effect of esfenvalerate in Bombina bombina on malformation, growth inhibition and mortality. Mean of 3 experiments.
Effekten af procloraz på Bombina bombina illustreret ved sublethale defekter, væksthæmning og dødelighed. Middeltal af 3 forsøg.
| Concentrations |
Malformation ( in procent) |
Growth inhibition (in m.m.) |
Mortality (in procent) |
| Control |
1.5 (S.D. = 0.7) |
9.5 (S.D. = 0.1) |
1.6 (S.D. = 2.4) |
| DMSO control |
1.7 (S.D. = 0.9) |
9.2 (S.D. = 0.1) |
1.0 (S.D. = 1.4) |
| 1.0 µg/l |
2.0 (S.D. = 1.6) |
9.3 (S.D. = 0.2) |
0.0 (S.D. = 0.0) |
| 2.5 µg/l |
2.3 (S.D. = 1.2) |
9.2 (S.D. = 0.1) |
0.0 (S.D. = 0.0) |
| 5.0 µg/l |
8.2 (S.D. = 1.0) |
9.4 (S.D. = 0.1) |
0.7 (S.D. = 0.9) |
| 10.0 µg/l |
11.7 (S.D. = 2.1) |
9.3 (S.D. = 0.2) |
0.3 (S.D. = 0.5) |
| 50.0 µg/l |
67.3 (S.D. = 9.2) |
8.9 (S.D. = 0,2) |
0.0 (S.D. = 0.0) |
| 100 µg/l |
100.0 (S.D. = 0.0) |
8.9 (S.D. = 0.2) |
0.0 (S.D. = 0.0) |
| 150 µg/l |
100.0 (S.D. = 0.0) |
9.2 (S.D. = 0.4) |
1.3 (S.D. = 0.5) |
Table A4.10
The effect of esfenvalerate in Bombina bombina on malformation, growth inhibition and mortality. The results are expressed as NOEC, LOEC and EC50/LC50 after 120 h of exposure.
Effekten af esfenvalerat på Bombina bombina illustreret ved sublethale defekter, væksthæmning og dødelighed. Resultaterne er udtrykt ved NOEC, LOEC og EC50/LC50 efter 120 timers eksponering.
| |
Malformations |
| NOEC |
LOEC |
EC50 |
| Means of 3 experiments |
2.5 µg/l |
5.0 µg/l |
29.3g/l |
| Standard deviations |
0.0 |
0.0 |
4.2 |
| The 95% confidence limits |
- |
- |
24.6- 34.0 |
| |
Growth inhibition |
| NOEC |
LOEC |
EC50 |
| Means of 3 experiments |
150 µg/l |
150 µg/l> |
150 µg/l> |
| Standard deviations |
0.0 |
0.0 |
0.0 |
| The 95% confidence limits |
- |
- |
- |
| |
Mortality |
| NOEC |
LOEC |
LC50 |
| Means of 3 experiments |
150g/l |
150 µg/l> |
150 µg/l> |
| Standard deviations |
0.0 |
0.0 |
0.0 |
| The 95% confidence limits |
- |
- |
- |
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Version 1.0 September 2004, © Danish Environmental Protection Agency
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