Effects of copper on Enteromorpha flexuosa (Chlorophyta) in vitro

Enteromorpha species are widely used as biomonitors of copper (Cu) contamination in coastal waters, but the effects of Cu at the subcellular level and possible mechanisms of metal resistance are scarcely known. To contribute to the understanding of the Cu accumulation process in macroalgae species, we exposed adult individuals of Enteromorpha flexuosa to 50, 250, and 500 microg Cu/L in seawater for 5 days under controlled conditions. Thereafter, gross photosynthesis rate (GPR) and metal accumulation were measured. Conventional transmission electron microscopy (CTEM) and energy-dispersive X-ray analysis (EDXA) were used to study the metal accumulation process at the cellular level. Treatments with 250 and 500 microg Cu/L were observed to cause an inhibition of the GPR in the algae. Cu accumulation in samples exposed to 500 microgCu/L was 5284+/-561 microg Cu/g (dry wt), whereas in control samples (no Cu addition), accumulation was 9+/-1 microg/g. In cells of plants undergoing the 50 microg Cu/L treatment, the cytoplasm was clearly vacuolated. Thickening of cell walls and increase of relative number of starch granules and of lipid bodies were the main cellular changes observed in plants exposed to 250 and 500 microg Cu/L. EDXA of algae cells after 250 and 500 microg Cu/L exposure detected Cu mainly in vacuole precipitates. Cu was also detected in chloroplasts and in some epiphytic bacteria. It was concluded that E. flexuosa did not avoid penetration of Cu into the cytoplasm and consequently its toxic effects in concentrations of 250 and 500 microg Cu/L. Precipitates containing Cu in vacuoles should be related to a metal immobilization mechanism, minimizing the Cu toxicity for cells. The epiphytic bacteria can act as a biofilter diminishing the availability of free Cu(+2) for algae accumulation.