No abstract

The influence of copper(II) concentration and speciation on the growth of Amphidinium carterae in batch culture in a modified ESAW medium was evaluated by two different experimental designs: (i) copper(If) was added to the cultures 2 h after cell inoculation and (ii) copper( II ) had a 24 h pre-equilibration period in the medium before cell inoculation. To complement the biological data, the following information on the chemical/biological system was obtained: (a) computational speciation of the culture medium under chemical equilibrium conditions: (b) total and electrochemically labile copper concentrations in the culture medium during the toxicity experiments; and (c) cellular levels of copper. The labile fraction of copper was measured by potentiometric stripping analysis (PSA) at a deposition potential (Ed) of --0"3 V. At this E d the dissociation of Cu-EDTA complexes was negligible. In both kinds of experiments the effects of copper were more evident after 4 days than after 7 or 10 days of exposure. When compared with situation (ii) pronounced toxic effects were observed under situation (i): (1) A. carterae growth rate was affected at lower copper concentration and in a more acute way: (2) the cells were exposed to significantly higher levels of labile copper (2-to 3-fold more than in situation (ii) for 13"4 #M total copper) and (3) after 2 h of exposure to the metal the copper sorbed by the cells was about 2-fold higher. These results provide evidence for the importance of kinetic parameters in toxicity studies. Experimental copper lability together with speciation calculations provided additional evidence that the bioavailable copper and its cellular toxicity are directly related to the labile metal concentration. This is higher than but directly proportional to the initial free metal concentration in the medium, as was experimentally verified. PSA-derived copper lability provided a reasonable indicator of metal bioavailability to A. carterae in this synthetic seawater medium. A first response of A. carterae to copper toxicity is the release of the metal due to re-equilibration between copper bound to the cell surface and to the ligands in the medium and/or efflux. A different mechanism must subsequently be activated, allowing growth despite the relatively high cellular levels of copper observed.

The influence of copper(II) on some aspects of the cell physiology and biochemistry of the dinoflage/lates Amphidinium carterae and Prorocentrum micans growing exponentially was examined in batch cultures. The concentration of labile copper (free ion plus that present in labile complexes) in the cultures was estimated taking into account the pH of the medium at the moment of copper addition, and the chemical composition of the medium. The speciation of other metals was also considered. A. carterae and P. micans exposed to concentrations of labile copper of, respectively, 605 nM and 55 nM, which resulted from the addition of 1"58 #M (I00/.tg dm -3) of total copper, showed a decrease in the growth of the cultures. Related to the number of cells in the cultures, these labile copper concentrations correspond to, respectively, 3"80 and 19"8 fmol of dissolved copper per cell. Growth of the two species in lower copper concentrations was similar to the growth of the control cultures. Besides a decrease in the growth of the cultures, higher copper concentrations also induced changes in cell motility. At the two highest copper concentrations a decrease in the protein content, only studied in P. micans, was observed. In P. rnicans, lethality was achieved with a concentration of labile copper of about 3.16 #M (406 hnol dissolved copper per cell) [resulting from 15"8 #M (1000 #g dm -3) of total copper]; there was both an irreversible loss of cell motility and growth inhibition (over the 21 days considered) and a large reduction in protein. Both species showed a certain ability to recover from sublethal copper doses. Copper effects and the difference in sensitivity of the two dinoflagellates to the metal are discussed.

The activity of phenethyl alcohol (PEA) on Bacillus cereus, B. megaterium, and Streptococcus faecalis was studied by electron microscopy of thin sections and by the assay of intracellular K+ leakage. S. faecalis was unaffected by PEA at concentrations up to 0.5%, B. cereus was severely damaged by 0.5% PEA, and B. megaterium behaved intermediately. Important membrane ultrastructural alterations were observed in B. cereus cells treated with 0.5% PEA, namely the change in the geometry of the membrane profile from asymmetric to symmetric, the occurrence of prominent, complex mesosome-like structures, and membrane fracturing and solubilization. Protoplasts from B. megaterium were found to be quickly lysed by 0.5% PEA due to the disruption of the cytoplasmic membrane. The electron microscopic observations, together with the results of the study of the K+ efflux from B. cereus and B. megaterium, indicate that PEA primarily and directly damages the cytoplasmic membrane of sensitive bacteria. The breakdown of the permeability barrier probably is responsible for the observed bactericidal action of 0.5% PEA on B. cereus.

We investigated how Prorocentrum micans Ehrenberg, a planktonic dinoflagellate common in Portuguese coastal waters, is able to tolerate and recover from sublethal concentrations of copper(II). The experimental design simulated events in inshore waters, where P. micans is subjected to high levels of pollutants, including copper. Decrease in growth rate, induction of a growth lag phase, temporary loss of motility, and potassium leakage were the effects induced in P. micans cultures by 90 nM labile copper. A 10–20‐fold increase in cellular copper concentration was observed in toxicity experiments. Copper efflux (representing a 50% decrease in cellular metal content) was a short‐term tolerance mechanism. A 25‐kDa protein was detected after only 3 h of exposure to copper, but there was no evidence of phytochelatin synthesis. Ultracytochemical labeling of metals with the sulfide‐silver procedure showed that copper was associated with the thecal plates, starch grains, and, to a lesser extent, lipid droplets. High values affixation capacities and average conditional stability constants for copper binding by starch, amylopectin, and cellulose support the location of copper in thecal plates and starch grains. We conclude that P. micans responds rapidly to copper toxicity and has two tolerance mechanisms for copper: copper efflux and sequestration in polymeric substances.