Toxic Effects of Copper, Cadmium, and Methoxychlor Shown by Neutral Red Retention Assay in Two Species of Freshwater Molluscs

We used neutral red retention assay in lysosomes of digestive gland cells as an indicator for stress effects by the environmental contaminants Cu, Cd, and the pesticide methoxychlor in two freshwater molluscs, the unionid mussel, Elliptio complanata (Lightfoot) and the ramshorn snail Helisoma trivolvis (Say). Mussels and snails were exposed for 7 and 14 days to Cu and Cd each at nominal concentrations of 2.5 μg/L, 5.0 μg/L, and 10.0 μg/L, and to methoxychlor concentrations of 1.0 μg/L, 10.0μg/L, and 100.0 μg/L. Both mussels and snails exposed to Cu showed a significant increase in the percent of destabilized lysosomes compared with lab control and freshly-collected (field control) mussels at both 7 and 14 days exposure for all concentrations. Cdexposed mussels did not show a significant difference with either of the controls at 7 days, but at 14 days exposure, Cd significantly increased the percent of destabilized lysosomes at all concentrations compared to field control mussels. Compared to laboratory controls, Cd increased lysosomal destabilization at 5.0 μg/L and 10.0 μg/L. Snails exposed to Cd for 7 days had a significantly higher percentage of lysosomal destabilization than both lab and field controls but at 14 days, significant differences were only seen at the two highest Cd concentrations. Methoxychlor-exposed mussels showed no significant lysosomal destabilization at 7 days compared to controls. But at 14 days exposure, the pesticide increased the percentage of lysosomal destabilization at 10.0μg/L compared to lab controls, and increased at both 10.0μg/L and 100.0μg/L compared to field control mussels. Methoxychlor-exposed snails had a higher percentage of lysosomal destabilization at 7 and 14 days at all concentrations compared to both controls with the exception of the 1.0 μg/L -7 day exposure group. Snails were more sensitive to Cd and to methoxychlor than were mussels possibly because they lack an operculum and are thus completely exposed to the environment. The lowest observed effect concentration (LOEC) for Cd was 2.5 μg/L (14 days) for Elliptio and 2.5 μg/L (7 and 14 days) for Helisoma. For methoxychlor, the LOEC was 10.0 μg/L (14 days) for Elliptio and 10.0 and 1.0 μg/L (7 and 14 days, respectively) for Helisoma. The LOEC for Cu was 2.5 μg/L (7 and 14 day exposure) for both Elliptio and Helisoma. These results show that lysosomal destabilization as indicated by neutral red retention is a reliable indicator of stress from heavy metals and a pesticide in freshwater molluscs, including a taxon that is endangered or threatened in North America.