After each exposure period, fish were removed from the aquaria
and immediately anesthetized with benzocaine (0.1 g L−1) and
blood samples were collected from the caudal vein using a heparinized
plastic syringe. Blood was used for the determination
of the frequency of micronuclei (MN) and other erythrocytic
nuclear abnormalities (ENA) and a blood sub-sample (10 L) were
stored in microtubes (1.5 mL) with 700 L of PBS (NaCl 126.6 mM,
KCL 4.8 mM, CaCl2 1.5 mM, NaHCO3 3.7 mM, Na2HPO4 8.9 mM,
NaH2PO4 2.9 mM) and kept on ice for the comet assay. Thereafter
animals were euthanized by medullar section for gills and liver
removal. Immediately after excision the gills and liver were carefully
washed with PBS and gill filaments and part of the liver were
transferred to microtubes for the cellular dissociation to be used
in the comet assay. Another part of the liver was transferred to
criotubes and kept frozen (−80 ◦C) until metallothionein analysis.