Biomarkers of waterborne copper exposure in the guppy Poecilia vivipara acclimated to salt water.

The responses of a large suite of biochemical and genetic parameters were evaluated in tissues (liver, gills, muscle and erythrocytes) of the estuarine guppy Poecilia vivipara exposed to waterborne copper in salt water (salinity 24 ppt). Activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione reductase, and glutathione S-transferase), metallothionein-like protein concentration, reactive oxygen species (ROS) content, antioxidant capacity against peroxyl radicals (ACAP), and lipid peroxidation (LPO) were evaluated in liver, gills, and muscle. Comet assay score and nuclear abnormalities and micronucleated cell frequency were analyzed in peripheral erythrocytes. The responses of these parameters were evaluated in fish exposed (96 h) to environmentally relevant copper concentrations (5, 9 and 20 µg L⁻¹). In control and copper-exposed fish, no mortality was observed over the experimental period. Almost all biochemical and genetic parameters proved to be affected by waterborne copper exposure. However, the response of catalase activity in liver, ROS, ACAP and LPO in muscle, gills and liver, and DNA damages in erythrocytes clearly showed to be dependent on copper concentration in salt water. Therefore, the use of these parameters could be of relevance in the scope of biomonitoring programs in salt water environments contaminated with copper.

Acute toxicity, accumulation and tissue distribution of copper in the blue crab Callinectes sapidus acclimated to different salinities: in vivo and in vitro studies.

In vivo and in vitro studies were performed to evaluate acute toxicity, organ-specific distribution, and tissue accumulation of copper in Callinectes sapidus acclimated to two different experimental salinities (2 and 30 ppt). Blue crabs were quite tolerant to copper. Acute dissolved copper toxicity (96-h LC(50) and its corresponding 95% confident interval) was higher at salinity 2 ppt (5.3 (3.50-8.05) µM Cu) than at 30 ppt (53.0 (27.39-102.52) µM Cu). The difference between salinities can be completely explained based on the water chemistry because it disappeared when 96-h LC(50) values were expressed as the free Cu(2+) ion (3.1 (1.93-4.95) µM free Cu at 2 ppt versus 5.6 (2.33-13.37) µM free Cu at 30 ppt) or the Cu(2+) activity (1.4 (0.88-2.26) µM Cu activity at 2 ppt versus 1.7 (0.71-4.07) µM Cu activity at 30 ppt). The relationships between gill Cu burden and % mortality were very similar at 2 and 30 ppt, in accord with the Biotic Ligand Model. In vivo experiments showed that copper concentration in the hemolymph is not dependent on metal concentration in the surrounding medium at either experimental salinity. They also showed that copper flux into the gills is higher than into other tissues analyzed, and that anterior and posterior gills are similarly important sites of copper accumulation at both experimental salinities. In vitro experiments with isolated-perfused gills showed that there is a positive relationship between copper accumulation in this tissue and the metal concentration in the incubation media for both anterior and posterior gills. A similar result was observed at both low and high salinities. Furthermore, in vitro experiments showed that copper accumulation in posterior gills is also positively and strongly dependent on the incubation time with copper. Gill copper accumulation occurred at a lower rate in the first 2h of metal exposure, increasing markedly after this "steady-state" period. This finding was corroborated by a significant increase in copper influx to the gill perfusate (corresponding to crab hemolymph) after this time, measured using (64)Cu. In vivo, after uptake from solution, (64)Cu was primarily accumulated in the gills and the rest of the body rather than in the hemolymph, hepatopancreas, or other internal tissues. Overall, the present findings indicate that gills are a key target organ for copper accumulation, as well as an important biological barrier against the excessive uptake of copper into the hemolymph and the subsequent distribution of this metal to internal organs of the blue crab.