Interactions of algal ligands, metal complexation and availability, and cell responses of the diatom Ditylum brightwellii with a gradual increase in copper.

A continuous culture experiment was conducted to study interactions between copper-binding ligands released by light-limited Ditylum brightwellii, and toxic effects of Cu on this diatom. Over 6 months, the Cu concentration in the medium has been increased in seven steps (3-173 nM). At each Cu addition, Cu speciation, characteristics of Cu sorption to cellular binding sites, and cell characteristics were determined. Physiological effects of Cu were studied, using indicators for metal detoxification (thiols) and lipid peroxidation (malondialdehyde). Minor amounts of Cu (<1.4%) were chelated by a minimum amount of EDTA (57 nM), required to maintain a stable long-term continuous culture. The responses of D. brightwellii to Cu were monitored. (1) From 3 to 47 nM added Cu, decreasing pools of glutathione, increasing malondialdehyde contents, an increased release of lipophilic ligands, and cell lysis indicated the enhancement of lipid peroxidation. (2) From 47 to 94 nM Cu, a 16-fold increase in high-affinity (strong) hydrophilic ligands was measured (conditional stability constants K' approximately 10(12)) that complexed most Cu (maximum 97%); sexual reproduction was stimulated and cell volumes increased. (3) From 126 nM Cu, glutathione pools increased again, whereas cell division rates decreased slightly. (4) At 142 nM Cu, the number of lysed cells reached a maximum, as did the production of lipophilic compounds that complexed approximately 2% Cu. As the binding sites of the strong ligands became Cu-saturated above 142 nM Cu, larger amounts of Cu were bound to low-affinity (weak) dissolved ligands (3-30%) and cellular binding sites (0.2-2.5%). Probably due to saturation of organic complexes at 142 nM Cu, the MINEQL-calculated Cu2+ concentrations increased markedly; pCu values decreased from >11 to approximately 10; division rates were further inhibited; gamma-glutamylcysteine (phytochelatin precursor) was produced. (5) At 157 nM Cu, phytochelatin synthesis started, and Cu-sorption capacities (cell walls and internal binding sites) increased. (6) At 173 nM Cu, the phytochelatin pool sizes and the number of cellular Cu-binding sites increased further. These results suggest that ligands released by a dense bloom of D. brightwellii, either by active excretion or lysis, would have lower affinities for Cu (K' approximately 10(9)-10(12)) and moderate the availability of Cu less effectively than ligands in natural environments (10(13)-10(14)). In this diatom, the concurring release of ligands, enhanced malondialdehyde production, increasing numbers of presexual cells and cell enlargement may serve as early-warning signals for Cu toxicity, rather than metal-specific phytochelatins that appeared at a stage when cell division was already clearly inhibited.

Interaction of toxic trace metals and mechanisms of detoxification in the planktonic diatoms Ditylum brightwellii and Thalassiosira pseudonana.

Effects of cadmium (10 nM), copper (80 nM) and zinc (150 nM) additions were studied in the marine diatom Ditylum brightwellii and the riverine diatom Thalassiosira pseudonana. Defense against oxidative stress via cellular thiol (SH) pools and superoxide dismutase (SOD) activation, detoxification via phytochelatins and cell damage were monitored in metal-exposed exponential-phase cells and controls, grown in estuarine medium. Total SH and reduced+oxidized glutathione (GSH+GSSG) in T. pseudonana were much higher than in D. brightwellii. In T. pseudonana, total SH and GSH decreased at 322 nM Zn, and GSH increased at 80 nM Cu but decreased at 119 nM Cu. GSH:GSSG ratios were low, while phytochelatins were not detectable in metal-exposed D. brightwellii. Cd-exposed T. pseudonana made more phytochelatins than Cu-exposed cells, and in different proportions. At 322 nM Zn, SOD activity decreased in T. pseudonana. Zn caused a major, and Cu a minor increase of SOD activity in D. brightwellii; inhibition of photosynthesis was observed in Cu-exposed D. brightwellii, probably due to oxidative damage. The C:N ratios were higher and protein contents lower in Cu-exposed cells of both species, which might indicate excretion due to a loss of cell membrane integrity. From these results, it is hypothesized that T. pseudonana has evolved an effective detoxification mechanism as a result of a more severe exposure to toxic metals in rivers and estuaries. In contrast, D. brightwellii, a marine-estuarine species, cannot adjust well to metal exposure. Its poor defense against metal toxicity was marked by low SH-contents.