Supplementary Material s68

Supplementary material

Enchytraeid Reproduction TestPLUS - hatching, growth and full life cycle test – an optional multi-endpoint test with Enchytraeus crypticus

Rita C. Bicho*, Fátima C. F. Santos, Micael F. M. Gonçalves, Amadeu M.V. M. Soares,

Mónica J.B. Amorim

Departamento de Biologia & CESAM, Universidade de Aveiro, 3810-193 Aveiro,

Portugal

*Corresponding author: [email protected]

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.

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)

Table S2 Results in terms of length of organisms from the full life cycle test with Cd

(average ± standard error;), including days 11, 25 and 46 days after cocoon deposition.

From each replicate a subsample of 20 organisms were measured (N=20 per replicate).

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

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

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

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.

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.

Dunn´s *p < 0.001.

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

2 Fig. S1 Schematic representation of the proposed test design for Hatching, Growth and

Full Life Cycle Test for Enchytraeus crypticus. Scheme includes the sampling days

(signed by black arrows) required per endpoint (hatching success, growth, maturity status, survival and reproduction), proposal of minimum replicates/treatment.

3 A FLCt B Gt

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 ( (

model fitted

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

time (days) time (days)

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))).

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.