Online Resource 1.
Metallothionein gene expression differs in earthworm populations with different exposure history
Journal: Ecotoxicology
Mustonen, M.,a Haimi, J.,a Väisänen, A.b and Knott, K. E.a
aDepartment of Biological and Environmental Science, University of Jyväskylä, P.O. Box 35, 40014, Jyväskylä, Finland
bDepartment of Chemistry, University of Jyväskylä, P.O. Box 35, 40014, Jyväskylä, Finland
Email of corresponding author:
Table 1. The intended and measured concentrations of Cu and Zn in soil used in the exposure experiments.
Treatment* / Intended concentration mg/kg / Measured concentration mg/kg**Cu low / 50 / 56.42
Cu medium / 100 / 118.5
Cu high / 200 / 181.9
Zn low / 75 / 131.8
Zn medium / 150 / 172.1
Zn high / 300 / 293.1
* Control soil had mean Cu concentration 9.96mg/kg and mean Zn concentration 42.67mg/kg.
Cu exposed:
Table 2. Effects of site (contaminated vs. uncontaminated), Cu exposure concentration, exposure time and their interactions on metallothionein expression in D. octaedra (Three-way ANOVA).
Source of variation / df / SS / MS / F / PSite / 1 / 69.31 / 69.31 / 47.51 / 0.001
Concentration / 3 / 3.714 / 1.238 / 0.849 / 0.472
Exposure time / 2 / 19.32 / 9.658 / 6.620 / 0.002
Site x Concentration / 3 / 4.127 / 1.376 / 0.943 / 0.425
Site x Exposure time / 2 / 17.00 / 8.500 / 5.826 / 0.005
Concentration x Exposure time / 6 / 9.136 / 1.523 / 1.044 / 0.405
Site x Conc. x Exposure time / 6 / 4.815 / 0.803 / 0.550 / 0.768
Table 3. Pairwise comparisons (post hoc LSD) of exposure times after three-way ANOVA on metallothionein expression of Cu exposed D. octaedra earthworms.
I / J / Difference (I-J) / P7 / 14 / 0.4667 / 0.127
14 / 28 / -1.4295 / <0.001
7 / 28 / -0.9628 / 0.003
Table 4. Effects of site, exposure concentration and their interaction on metallothionein expression of Cu exposed D. octaedra earthworms with exposure times separated (Two-way ANOVA).
Days of exposure / Source of variation / df / SS / MS / F / P7 / Site / 1 / 12.48 / 12.48 / 11.52 / 0.003
Concentration / 3 / 3.940 / 1.313 / 1.213 / 0.329
Site x Concentration / 3 / 0.995 / 0.332 / 0.306 / 0.820
14 / Site / 1 / 68.95 / 68.95 / 46.10 / 0.001
Concentration / 3 / 0.939 / 0.313 / 0.209 / 0.889
Site x Concentration / 3 / 2.463 / 0.821 / 0.549 / 0.653
28 / Site / 1 / 7.371 / 7.371 / 4.114 / 0.055
Concentration / 3 / 7.714 / 2.571 / 1.435 / 0.259
Site x Concentration / 3 / 5.714 / 1.905 / 1.063 / 0.385
Table 5. Effects of Cu exposure concentration, exposure time and their interaction on metallothionein expression of D. octaedra earthworms from the contaminated and uncontaminated sites (Two-way ANOVA).
Site / Source of variation / df / SS / MS / F / PContaminated / Concentration / 3 / 1.614 / 0.538 / 0.266 / 0.849
Exposure time / 2 / 3.685 / 1.842 / 0.910 / 0.413
Concentration x Exposure time / 6 / 6.301 / 1.050 / 0.519 / 0.790
Uncontaminated / Concentration / 3 / 6.816 / 2.272 / 2.251 / 0.098
Exposure time / 2 / 35.53 / 17.77 / 17.60 / <0.001
Concentration x Exposure time / 6 / 8.261 / 1.377 / 1.364 / 0.253
Table 6. Pairwise comparisons (post hoc LSD) of exposure times after two-way ANOVA on metallothionein expression of Cu exposed D. octaedra earthworms from the uncontaminated site.
I / J / Difference (I-J) / Sig.7 / 14 / 1.0741 / 0.002
14 / 28 / -2.1657 / <0.001
7 / 28 / -1.0915 / 0.005
Table 7. Effect of exposure time on metallothionein expression of Cu exposed D. octaedra earthworms from the uncontaminated site with exposure concentrations separated (One-way ANOVA).
Exposure level / df / SS / MS / F / SigControl / 2 / 1.011 / 0.506 / 0.378 / 0.694
Low / 2 / 7.471 / 3.736 / 3.734 / 0.066
Medium / 2 / 24.95 / 12.47 / 11.86 / 0.002
High / 2 / 12.56 / 6.282 / 10.40 / 0.005
Table 8. Pairwise comparisons (post hoc LSD) of exposure times on medium and high treatments after one-way ANOVA on metallothionein expression of Cu exposed D. octaedra earthworms from the uncontaminated site.
Exposure level / I / J / Difference (I-J) / Sig.Medium / 7 / 14 / 1.6692 / 0.026
14 / 28 / -3.3432 / 0.001
7 / 28 / -1.6740 / 0.033
High / 7 / 14 / 1.3766 / 0.038
14 / 28 / -2.3545 / 0.001
7 / 28 / -0.9779 / 0.134
Table 9. Effects of site, exposure time and their interaction on metallothionein expression of Cu exposed D. octaedra earthworms with exposure concentrations separated (Two-way ANOVA).
Exposure level / Source of variation / df / SS / MS / F / PControl / Site / 1 / 13.28 / 13.28 / 6.671 / 0.019
Exposure time / 2 / 0.606 / 0.303 / 0.152 / 0.860
Site x Exposure time / 2 / 1.297 / 0.649 / 0.326 / 0.726
Low / Site / 1 / 31.05 / 31.05 / 26.32 / 0.001
Exposure time / 2 / 9.142 / 4.571 / 3.875 / 0.044
Site x Exposure time / 2 / 3.798 / 1.899 / 1.610 / 0.233
Medium / Site / 1 / 12.08 / 12.08 / 9.296 / 0.006
Exposure time / 2 / 16.01 / 8.005 / 6.162 / 0.008
Site x Exposure time / 2 / 9.246 / 4.623 / 3.559 / 0.048
High / Site / 1 / 14.82 / 14.82 / 10.85 / 0.004
Exposure time / 2 / 3.786 / 1.893 / 1.385 / 0.276
Site x Exposure time / 2 / 8.400 / 4.200 / 3.074 / 0.071
Table 10. Pairwise comparisons (post hoc LSD) of exposure times on control, low and medium treatments after two-way ANOVA on metallothionein expression of Cu exposed D. octaedra earthworms.
Exposure level / I / J / Difference (I-J) / Sig.Control / 7 / 14 / -0.2726 / 0.700
14 / 28 / -0.3491 / 0.638
7 / 28 / -0.6217 / 0.439
Low / 7 / 14 / 0.2333 / 0.694
14 / 28 / -2.0969 / 0.003
7 / 28 / -1.8637 / 0.006
Medium / 7 / 14 / 0.7514 / 0.177
14 / 28 / -2.0901 / 0.001
7 / 28 / -1.3387 / 0.025
Zn exposed:
Table 11. Effects of site, exposure concentration, exposure time and their interactions on metallothionein expression in Zn exposed D. octaedra earthworms (Three-way ANOVA).
Source of variation / df / SS / MS / F / PSite / 1 / 82.92 / 82.92 / 56.87 / <0.001
Concentration / 3 / 31.11 / 10.37 / 7.112 / <0.001
Exposure time / 2 / 22.28 / 11.14 / 7.642 / 0.001
Site x Concentration / 3 / 8.608 / 2.869 / 1.968 / 0.126
Site x Exposure time / 2 / 7.885 / 3.943 / 2.704 / 0.073
Concentration x Exposure time / 6 / 17.062 / 2.844 / 1.950 / 0.083
Site x Conc. x Exposure time / 6 / 17.216 / 2.869 / 1.968 / 0.080
Table 12. Pairwise comparisons (post hoc LSD) of exposure concentrations after three-way ANOVA on metallothionein expression of Zn exposed D. octaedra earthworms.
I / J / Difference (I-J) / Sig.Control / Low / 1.2176 / 0.001
Medium / 1.3354 / <0.001
High / 1.3320 / <0.001
Low / Medium / 0.1179 / 0.726
Medium / High / -0.0034 / 0.992
High / Low / -0.1144 / 0.726
Table 13. Pairwise comparisons (post hoc LSD) of exposure times after three-way ANOVA on metallothionein expression of Zn exposed D. octaedra earthworms.
I / J / Difference (I-J) / Sig.7 / 14 / -0.3535 / 0.225
14 / 28 / -0.8830 / 0.004
7 / 28 / -1.2365 / <0.001
Table 14. Effects of site, exposure concentration and their interaction on metallothionein expression of Zn exposed D. octaedra earthworms with exposure times separated (Two-way ANOVA).
Exposure time / Source of variation / df / SS / MS / F / P7 / Site / 1 / 32.23 / 32.23 / 20.00 / 0.001
Concentration / 3 / 17.36 / 5.787 / 3.592 / 0.027
Site x Concentration / 3 / 7.410 / 2.470 / 1.533 / 0.229
14 / Site / 1 / 53.56 / 53.56 / 39.73 / 0.001
Concentration / 3 / 29.88 / 9.959 / 7.387 / 0.001
Site x Concentration / 3 / 1.471 / 0.490 / 0.364 / 0.780
28 / Site / 1 / 8.783 / 8.783 / 6.182 / 0.020
Concentration / 3 / 3.419 / 1.140 / 0.802 / 0.505
Site x Concentration / 3 / 16.46 / 5.487 / 3.863 / 0.022
Table 15. Pairwise comparisons (post hoc LSD) of exposure concentrations after two-way ANOVA on metallothionein expression of Zn exposed D. octaedra earthworms.
Exposure time / I / J / Difference (I-J) / Sig.7 / Control / Low / 0.9898 / 0.134
Medium / 2.0600* / 0.003
High / 1.6083* / 0.018
Low / Medium / 1.0702 / 0.085
Medium / High / -0.4517 / 0.457
High / Low / -0.6184 / 0.311
14 / Control / Low / 2.0438 / 0.001
Medium / 0.7658 / 0.162
High / 2.0238 / 0.001
Low / Medium / -1.2781 / 0.027
Medium / High / 1.2580 / 0.034
High / Low / 0.0201 / 0.972
Table 16. Effects of exposure concentration, exposure time and their interaction on metallothionein expression of Zn exposed D. octaedra earthworms with sites separated (Two-way ANOVA).
Site / Source of variation / df / SS / MS / F / PContaminated / Concentration / 3 / 15.01 / 5.003 / 3.500 / 0.025
Exposure time / 2 / 2.283 / 1.142 / 0.799 / 0.458
Concentration x Exposure time / 6 / 13.59 / 2.265 / 1.584 / 0.179
Uncontaminated / Concentration / 3 / 26.24 / 8.747 / 5.894 / 0.002
Exposure time / 2 / 28.01 / 14.00 / 9.437 / <0.001
Concentration x Exposure time / 6 / 22.57 / 3.761 / 2.535 / 0.035
Table 17. Pairwise comparisons (post hoc LSD) of exposure concentrations after two-way ANOVA on metallothionein expression of Zn exposed D. octaedra earthworms from the uncontaminated site.
I / J / Difference (I-J) / Sig.Control / Low / 1.8435 / <0.001
Medium / 1.5532 / 0.003
High / 1.2831 / 0.009
Low / Medium / -0.2903 / 0.548
Medium / High / -0.2701 / 0.576
High / Low / 0.5604 / 0.231
Table 18. Pairwise comparisons (post hoc LSD) of exposure times after two-way ANOVA on metallothionein expression of Zn exposed D. octaedra earthworms from the uncontaminated site.
I / J / Difference (I-J) / Sig.7 / 14 / 0.1277 / 0.752
14 / 28 / -1.5098 / 0.001
7 / 28 / -1.3821 / 0.002
Table 19. Effect of exposure time on metallothionein expression of Zn exposed D. octaedra earthworms from the contaminated and uncontaminated site with exposure concentrations separated (One-way ANOVA).
Site / Concentration / df / SS / MS / F / SigContaminated / Control / 2 / 0.748 / 0.374 / 0.128 / 0.882
Zn 75mg/kg (Low) / 2 / 7.193 / 3.596 / 4.624 / 0.035
Zn 150mg/kg (Medium) / 2 / 4.345 / 2.172 / 2.287 / 0.157
Zn 300mg/kg (High) / 2 / 5.145 / 2.572 / 1.682 / 0.235
Uncontaminated / Control / 2 / 1.011 / 0.506 / 0.378 / 0.694
Zn 75mg/kg (Low) / 2 / 8.709 / 4.355 / 3.198 / 0.080
Zn 150mg/kg (Medium) / 2 / 27.46 / 13.73 / 9.051 / 0.007
Zn 300mg/kg (High) / 2 / 13.78 / 6.891 / 4.021 / 0.049
Metal body content of earthworms:
Table 20. Effects of site, exposure concentration, exposure time and their interactions on Cu content in Cu exposed D. octaedra earthworms (Three-way ANOVA).
Source of variation / df / SS / MS / F / PSite / 1 / 1.589 / 1.589 / 4.552 / 0.036
Concentration / 3 / 2.862 / 0.954 / 2.733 / 0.050
Exposure time / 2 / 0.221 / 0.111 / 0.317 / 0.729
Site x Concentration / 3 / 2.159 / 0.720 / 2.062 / 0.113
Site x Exposure time / 2 / 1.032 / 0.516 / 1.479 / 0.235
Concentration x Exposure time / 6 / 1.303 / 0.217 / 0.622 / 0.712
Site x Conc. x Exposure time / 6 / 1.885 / 0.314 / 0.900 / 0.500
Table 21. Effects of site, exposure concentration, exposure time and their interactions on Zn content in Zn exposed D. octaedra earthworms (Three-way ANOVA).
Source of variation / df / SS / MS / F / PSite / 1 / 0.049 / 0.049 / 0.243 / 0.623
Concentration / 3 / 0.261 / 0.087 / 0.434 / 0.729
Exposure time / 2 / 0.108 / 0.054 / 0.270 / 0.764
Site x Concentration / 3 / 0.436 / 0.145 / 0.726 / 0.539
Site x Exposure time / 2 / 0.266 / 0.133 / 0.664 / 0.518
Concentration x Exposure time / 6 / 1.797 / 0.300 / 1.496 / 0.191
Site x Conc. x Exposure time / 6 / 0.805 / 0.134 / 0.670 / 0.674