IMIS

Mature individuals deposited in PVC tanks with Filtered Sea Water (FSW, 35-37 promille). Cyclic changes of water temperature, from 14°C to 20°C to induce spawning. Spawning individuals put into separate plastic containers to collect sperm and oocytes. After fertilization, incubation of fertilized eggs at a density of 40 individuals/mL. Periodical sampling to collect fertilized egg, trochophore larval stage (18-20h post-fertilization) and 48 and 72h-old D-larval stage for experiments. Cryoprotecting agent solutions prepared at twice the final concentration for the experiments. For toxicity tests, preparation of increasing concentrations (from 1 to 6 M) of Ethylene-Glycol (EG), Propylene-Glycol (PG), Dimethyl-Sulfoxide (Me2SO) and Glycerol (GLY) prepared in FSW. For cryopreservation experiments, 10% (v/v) EG + 0.4M (w/v) trehalose (TRE). Three replicates were assayed for each CPA concentration and control trials. Cryoprotectant solutions added 1:1 to a FSW mL with 400-600 clam individuals in one step. 15 minutes of exposure at room temperature (18-20°C). Samples diluted with FSW 1:1. Then, incubation in 20 mL of clean FSW at 18°C until D-larval stage was reached for exposed fertilized eggs and trochophores, 48h of incubation in the case of D-larval stage. After that, cells fixed with formalin for normal and abnormal D-larval counts. Obtention of normal and abnormal larval precentages, then arcsin-square transformation of data. For toxicity results, statistical analysis following ANOVA-one-way and calculations of NOEC and LOEC levels by post-hoc Dunnet test. For cryopreservation experiments, Bonferroni post-hoc test, assuming homogeneity of variances.