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In the present study, we used fresh preparations of mixed-cell populations to evaluate accumulation and toxicity of dissolved copper (1-100 microM) in isolated cells from posterior gills and hepatopancreas of the blue crab (Callinectes sapidus). For both gill and hepatopancreatic cells, significant increases in copper accumulation were observed after exposure to 50 or 100 microM copper. In gill cells, a linear increase in copper accumulation was observed over time. In hepatopancreatic cells, a maximum level of copper accumulation was achieved after 1 h of exposure, remaining unchanged up to 6 h. After 6 h of exposure, copper content in gill cells was 6.6-fold higher than that in hepatopancreatic cells. In both cell types, copper accumulation always followed a linear relationship with copper concentration in the incubation medium. Significant decreases in cell viability were observed after exposure to either 10 microM copper (gill cells) or 100 microM copper (gill and hepatopancreatic cells). Furthermore, an exponential rise to maximum-type relationship was observed between copper accumulation and toxicity in gill cells. Altogether, these findings indicate that the premise behind the biotic ligand model (BLM) approach is verified in isolated cells from posterior gills of the blue crab (i.e., toxicity is driven by copper accumulation in the biotic ligand, the gill cell). Therefore, these cells can be used as a model for the development of an in vitro BLM version for marine conditions. Isolated cells from the hepatopancreas, however, could be used as a model to better understand the mechanism of copper tolerance at a cellular level in crustaceans.

Imposex incidence, organotin tissue levels, and sex steroid (free and esterified testosterone and estradiol) levels were assessed in Stramonita haemastoma from Babitonga Bay (Santa Catarina State, Southern Brazil). The imposex levels showed a reduction when compared to a previous evaluation performed in the same area. In spite of that, the detected imposex incidence indicated the occurrence of tributyltin (TBT) inputs that were still able to produce endocrine disruption in local gastropods. In addition, a high level of organotins was observed in tissues of imposexed females. These females also showed a hormonal imbalance, especially in the total testosterone/total estradiol ratio. These findings obtained under realistic field conditions suggest that the steroid pathway could be responsible by the imposex induction after exposure to TBT. In this case, measurements of sex steroid levels can be an additional evidence for monitoring sites and impose affected gastropod populations.

The estuarine crab Neohelice granulata was maintained under control condition or exposed to sublethal concentrations of dissolved Ni (measured: 128 and 1010μg/L) for 96h at different salinities (2 and 30ppt). After metal exposure, whole-body oxygen consumption was measured and tissue (hemolymph, gills, hepatopancreas and muscle) samples were collected. Control crabs acclimated to 2ppt salinity showed lower hemolymph concentrations of Na (33%), Mg (19%) and K (30%), as well as increased LPO levels in anterior gills (379%), posterior gills (457%) and hepatopancreas (35%) with respect to those acclimated to 30ppt salinity. In crabs acclimated to 2ppt salinity, Ni exposure increased whole-body oxygen consumption (75%), hemolymph K concentration (52%), hemolymph (135%) and hepatopancreas (62%) LDH activity. Also, it reduced hemolymph Cl concentration (16%) and muscle LDH activity (33%). In crabs acclimated to 30ppt salinity, Ni exposure increased LDH activity in hemolymph (195%), hepatopancreas (126%) and muscle (53%), as well as hemolymph osmolality (10%), Cl (26%) and Ca (20%) concentration. It also reduced hepatopancreas lipid peroxidation (20%) and hemolymph Mg (29%) and K (31%) concentration. These findings indicate that N. granulata is hyper-osmoregulating in 2ppt salinity and hypo-regulating in 30ppt salinity, showing adjustments of hemolymph ionic composition and metabolic rates, with consequent higher oxidative damage to lipids in low salinity (2ppt). Ni effects are associated with metabolic (aerobic and anaerobic) disturbances in crabs acclimated to 2ppt salinity, while osmotic and ionoregulatory disturbances were more evident in crabs acclimated to 30ppt salinity.

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