Effects of copper exposure on the energy metabolism in juveniles of the marine clam Mesodesma mactroides.

In freshwater osmoregulating mollusks, Cu can cause toxicity by inducing ionoregulatory disturbances. In mussels, it inhibits the activity of key enzymes involved in Na(+) uptake and consequently induces ionic and osmotic disturbances. In snails, Cu induces disruption of the Ca(2+) homeostasis leading to effects in shell deposition and snail growth. However, the mechanisms involved in Cu toxicity in osmoconforming sweater mollusks remain unclear. Recent findings from our laboratory have suggested that Cu toxicity in marine invertebrates can be associated with both ionic and respiratory disturbances. In the present study, metabolic changes induced by waterborne Cu exposure were evaluated in the osmoconforming clam Mesodesma mactroides, a bivalve species widely distributed along the South American sandy beaches. Juvenile clams were kept under control conditions (no Cu addition in the water) or acutely (96h) exposed to Cu (96-h LC10=150µgL(-1)) in artificial seawater (30ppt). ATP, protein, lipid, glycogen and glucose contents were analyzed in gills, digestive gland, pedal muscle and hemolymph. Dinucleotide (NAD(+) and NADH) content was also analyzed in gills, digestive gland and pedal muscle while pyruvate and lactate content was determined in pedal muscle and hemolymph. In all tissues analyzed, Cu exposure did not affect ATP content and NAD(+)/NADH ratio, except in the hemolymph, where a decrease in ATP content was observed. These findings indicate that clam cells, except those from hemolymph, were able to maintain a constant level of free energy. A significant increase in total protein content was observed in the digestive gland, which could be a compensatory mechanism to counteract the higher level of protein oxidation previously observed in M. mactroides exposed to Cu under the same experimental conditions. Finally, reduced glucose content in the pedal muscle paralleled by increased lactate content in the pedal muscle and hemolymph was observed in Cu-exposed clams. Overall, these findings indicate that Cu exposure is leading to an increased reliance upon the anaerobic energy production to maintain the overall cellular ATP production in the clam M. mactroides.

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.