<p>Supplementary material</p><p>Enchytraeid Reproduction TestPLUS - hatching, growth and full life cycle test – an optional multi-endpoint test with Enchytraeus crypticus</p><p>Rita C. Bicho*, Fátima C. F. Santos, Micael F. M. Gonçalves, Amadeu M.V. M. Soares,</p><p>Mónica J.B. Amorim</p><p>Departamento de Biologia & CESAM, Universidade de Aveiro, 3810-193 Aveiro, </p><p>Portugal</p><p>*Corresponding author: [email protected]</p><p>Table S1: Results in terms of number of organisms (average ± standard error) and length measurements from the full life cycle test in control conditions samples during growth (days 14-18-22), maturity status (days 25-29) and reproduction (day 50), indicating survival and reproductive output.</p><p>Time No. juveniles No. adults length N (days) (mm) 14 32.3 ± 5.1 - 1.5 ± 0.02 94 18 32.0 ± 5.2 - 2.4 ± 0.05 98 22 35.0 ± 1.7 - 3.4 ± 0.06 104 25 - 29.3 ± 1.2 4.4 ± 0.05 90 29 - 32 ± 1.4 4.8 ± 0.05 98 50 924.6 ± 35 39 ± 2 5.19 ± 0.04 44 (from reproduction)</p><p>Table S2 Results in terms of length of organisms from the full life cycle test with Cd</p><p>(average ± standard error;), including days 11, 25 and 46 days after cocoon deposition.</p><p>From each replicate a subsample of 20 organisms were measured (N=20 per replicate). </p><p>Treatment juvenile N adult N juvenile adult N (mg Cd /kg) (mm) (mm) (mm) (mm) days 11 25 46 46 0 1.2±0.01 80 5.2±0.48 80 1.5±0.03 4.9±0.05 80 0.16 1.3±0.01 80 5.2±0.41 80 1.5±0.04 5.0±0.05 80</p><p>1 1.6 1.3±0.01 80 4.9±0.63 80 1.5±0.03 4.4±0.07 80 5 1.2±0.12 80 4.4±0.93 80 1.5±0.03 4.7±0.05 80 16 1.3 1 4.7±0.37 13 1.9±0.11 7.5±0.37 6a+27j</p><p>The results of the hatching test performed to compare cocoons with starting age between 1-2 and 1-3 days old in a limit test with 5 mg Cd/kg can be observed in table</p><p>S3. Significant differences were observed in terms of reduction in the number of juveniles hatched from cocoons with 1-2 days old compared to no effects for the 1-3 days old cocoons.</p><p>Table S3 Results in terms of number of juveniles (average ± standard error) for hatching test with Cd, comparing hatchability success of 1-2 and 1-3 days old cocoons.</p><p>Dunn´s *p < 0.001.</p><p>Cadmium No. juveniles (mg Cd /kg) 1-2 days 1-3days 0 37.2 ± 1.9 41.8 ± 3.7 5 16 ± 3.2* 41.8 ± 2.1</p><p>2 Fig. S1 Schematic representation of the proposed test design for Hatching, Growth and</p><p>Full Life Cycle Test for Enchytraeus crypticus. Scheme includes the sampling days</p><p>(signed by black arrows) required per endpoint (hatching success, growth, maturity status, survival and reproduction), proposal of minimum replicates/treatment. </p><p>3 A FLCt B Gt </p><p>8 8 0 mg/kg 7 7 1.6 mg/kg 6 6 5 mg/kg 16 mg/kg ) ) m 5 m 5 50 mg/kg m m ( (</p><p> model fitted</p><p> t 4 t 4 h h 95% confidence interval g g n 3 n 3 95% prediction interval e e l l 2 2 1 1 0 0 14 18 2225 29 50 14 18 22 25</p><p> time (days) time (days)</p><p>Fig. S2 Results of growth (length) for Enchytraeus crypticus in LUFA 2.2 standard natural soil from the A : full life cycle test (FLCt) in control conditions and B : growth test (Gt) when exposed to CdCl2 (0-1.6-5-16-50mg Cd/kg soil DW). All data points are plotted and black line represents the fit to sigmoid model (f= a/(1+exp(-(x-x0)/b))).</p><p>Model parameters for A: a=5.3; b= 4.5; x0=18.8. Model parameters for B: a=6.4; b=3.1; x0=16.7. </p><p>4</p>