Influence of phytoplankton on complexing capacity of seawater†

Differential Pulse Anodic Stripping Voltammetry (DPASV) was employed to measure Apparent Copper Complexing Capacity (L_t) and Stability Constants (K_s) of related cupric complexes of cell exudates of the phytoplanktonic alga Dunaliella salina. Three natural seawater samples collected 0.1, 2, 5 miles off the Tyrrhenian coast were used as culture medium. The measurements were carried out 1h and 7 days after the inoculum. Furthermore, on one culture the medium was passed through C₁₈‐SEP‐PAK cartridges in order to separate the cell exudates. The results show that Dunaliella salina influences the value of L_t but does not affect the value of K_s . The C₁₈SEP‐PAK retain only 50% of the ligands produced by the cells and capable of complexing copper.     

Mechanisms of copper tolerance in the marine fouling alga Ectocarpus siliculosus — Evidence for an exclusion mechanism

Copper-tolerant strains of the marine fouling alga Ectocarpus siliculosus (Dillw) Lyngb. have been recorded. The mechanisms of tolerance which may operate in a copper-tolerant strain were investigated by comparing the growth characteristics, copper content and extracellular organic production of tolerant and non-tolerant strains. Growth was measured by a wet-weight method. The copper content of dryashed cells was measured spectrophotometrically using tetraethylthiuram disulphide. The extracellular material is ultra-violet absorbing, and the production was measured by recording absorption at 265 nm. Evidence suggests that an exclusion mechanism is operating in the tolerant strain under investigation. The extracellular material, the cell wall or an associated organism make little or no contribution no this exclusion. Membrane and intracellular changes are believed to account for the tolerance in this strain of E. siliculosus.     

Effect of complexation by natural phytoplankton exudates on the accumulation of cadmium and copper by the haptophyceae Cricosphaera elongata

The accumulation of cadmium and copper by Cricosphaera elongata is determined in the absence and presence of natural phytoplankton exudates. The concentrations of metals accumulated increase with increasing concentrations of metals in the medium. Organic substances liberated into the culture medium by C. elongata are able to decrease the amounts of metals taken up.     

Physiological responses ofCalanus finmarchicus andMetridia longa (Copepoda: Calanoida) during the winter-spring transition

The effect of pH on the biological availability of copper to the brine shrimp Artemia franciscana was studied with acclimated and non-acclimated individuals for the pH range 5.5 to 8.5. A chemical speciation model was used to calculate the speciation of copper in a chemically-defined saline solution as a function of pH. The lipid solubility of inorganic copper species was determined in hexadecane/saline and octanol/saline extraction systems. Copper is absorbed across the gut epithelium and accumulation is proportional to time over a 120 min experimental period. The biological availability of copper decreases with a decrease in the pH of the salt solution and a concomitant increase of the cupric ion concentration. Acclimation to the experimental pH has a marked effect on the uptake process, which depends on the buffer used. There is no extraction of copper in hexadecane, but a small amount goes in octanol. There is, however, no direct relation between the accumulation of copper in the shrimps and the extraction of copper in octanol. Multiple regression of the accumulation rates for non-acclimated animals on the calculated copper species concentrations shows that much of the variation in accumulation rate with pH is explained when copper hydroxide and/or copper carbonate species are considered to be the biologically available forms. Alternatively, the observations can be interpreted as the result of competitive binding of protons and copper species for carrier systems.     

Acclimation and tolerance of Artemia salina and Ophryotrocha labronica to cooper sulphate

The brine shrimp Artemia salina L. and the polychaete worm Ophryotrocha labronica La Greca and Bacci were acclimated in sea water with copper sulphate at concentrations of 0.1, 0.05, and 0.025 ppm Cu⁺⁺, for 3 and 2 generations, respectively. Both adults and larvae of A. salina showed a greater tolerance to 1 ppm Cu⁺⁺ after acclimation compared to controls of the same age, although this tolerance diminished in successive generations. The acclimation effect was less marked in O. labronica. In both species, tolerance to 10 ppm Cu⁺⁺ upwards was not enhanced. Growth-rate inhibition and an adverse effect on reproduction was observed, in some instances in direct relationship to the acclimation concentration. It is suggested that, in A. salina, a certain tolerance to copper may be acquired through exposure to low concentrations.     

Mechanisms of copper tolerance by Armeria maritima in Dolfrwyong Bog,north Wales--initial studies

Preliminary field studies were carried out at Dolfrwynog Bog in July 2000. Replicate samples of water, Armeria maritima plants and the soils adhering to its roots were collected and analysed for copper. Concentrations of up to 6486 mg kg^–1 of copper in the soils were recorded. Accumulation of copper by the plant as expressed by concentration factors (CF) show that it is acting mainly as a copper excluder. Of the copper that is taken up, most of it is retained within the roots with very little being transported to the shoots of the plant. Moreover, a further possible mechanism of tolerance is exhibited by the excretion of copper through its decaying leaves. Towards the use of in vitro cultures to study the copper tolerance mechanisms in A. maritima a micropropagation protocol has been developed. The ex vitro plants have been rooted and established in compost.     

Interactions between two species of marine diatoms: Effects on their individual copper tolerance

The purpose of this study was to examine the effect of the interaction of two species of marine diatoms,Skeletonema costatum (Cleve) Greville andNitzschia thermalis (Ehrenberg) Auerswald, on their individual copper tolerances. The two species, obtained from stock cultures in 1989, were grown together at three copper concentrations (1, 4 and 5 × 10^–7 M added total copper). In the unialgal cultures that were used as controls, the two species grew as predicted from their tolerance tests. However, in mixed cultures,N. thermalis was the only species that exhibited growth, regardless of the copper concentration in the medium. Growth retardation ofS. costatum in the presence ofN. thermalis was attributed to an inhibitory exudate. The effect of the exudate appeared to have been temporary, as demonstrated by the extended lag phase and subsequent satisfactory exponential growth rate ofS. costatum. It is suggested that the exudate degraded within a period of 5 d (=lag phase) because exponential growth rate was resumed. It thus appears that the interaction between the two diatom species is more important in determining the survival ofS. costatum than its individual copper tolerance. This is not the case forN. thermalis. Such interactions would be unaccounted for in single-species toxicity tests. On the other hand, if they are known, prediction of how a community that includes these two species would respond to copper additions becomes possible.     

Do toxicity and accumulation of copper change during size reduction in the marine pennate diatom Haslea ostrearia?

 One of the best-known features of diatom biology is the reduction in mean cell size during vegetative multiplication by binary fission. We examined changes in copper toxicity and copper accumulation during cell-size reduction in Haslea ostrearia (Simonsen), a pennate diatom responsible for greening in oyster-ponds. We selected three strains with apical axes of different lengths: 40 μm (S40), 65 μm (S65) and 85 μm (S85). Each strain was grown separately in batch culture and exposed to a range of copper overloads (0 to 1.57 μM) that were added to the culture immediately after cell inoculation. Significant differences in sensitivity to copper were observed among the three strains. S85 exhibited highest sensitivity, followed by S40, while S65 displayed the highest tolerance. After 5 to 6 d exposure to 0.47 μM copper, chlorophyll a, carbohydrate, protein and lipid content per g dry weight had not changed in any of the three strains studied, except for a decrease of 16% in chlorophyll a in S85. At the end of the growth period with 0.47 μM copper, the amount of metal per unit surface area was similar for all strains, but the quantity of intracellular copper per g dry weight was lower in S65 cells than in S40 and S85 cells. Notable differences in the kinetics of both adsorbed and intracellular copper were observed between S40 and S85. Our results suggest that tolerance mechanisms may change during the vegetative life of H. ostrearia. The differential sensitivities of the strains suggest that copper pollution may alter the cell composition of natural populations of H. ostrearia by inducing selection for smaller cell size. Since auxosporulation results in the formation of larger cells with a higher sensitivity to copper, H. ostrearia could gradually disappear from copper-contaminated environments. In addition, by inducing smaller cell size, copper contamination would have an impact on filter-feeders such as oysters, whose diet is largely composed of diatoms. Received: 26 March 1999 / Accepted: 2 December 1999     

Effects of zinc stress on growth and antioxidant enzyme responses of Kandelia obovata seedlings

This study evaluated the tolerance and accumulation potential in the mangrove Kandelia obovata under moderate and high levels of external Zn. A greenhouse experiment was conducted to investigate the effects of a range of external Zn concentrations (0–400 mg L^?1) on the growth of this species by counting the lateral root number and measuring the root length, leaf area, and total dry biomass. We also determined the Zn accumulation in plant tissues. K. obovata survived with external Zn concentrations of up to 400 mg L^?1, although the excess metal resulted in a biomass reduction of 34%. A significant increase in antioxidant enzyme activities occurred in roots of plants under high-level Zn stress, suggesting that K. obovata seedlings could tolerate up to 200 mg L^?1 zinc treatment.     

Bioaccumulation and toxic effects of copper on growth and oxygen consumption by the postlarvae of Penaeus indicus

The aim of this study was to assess the toxic impact of copper on postlarvae (PL) of the penaeid shrimp Penaeus indicus. Tolerance, growth, oxygen consumption and metal accumulation were investigated in these PL on exposure to copper. Tolerance studies were conducted for 96 h to assess the tolerance limits of P. indicus PL exposed to different concentrations of copper using static renewal bioassay tests. Using the Probit method, the regression equation was calculated as Y=0.4899+2.3562 X, with a correlation coefficient of 0.9707. The 96 h LC₅₀ was 0.8204 ppm. The effect of sublethal (one-fifth of 96 h LC₅₀) copper on PL for short- and long-term exposures revealed a significant (p<0.05) decrease in the rate of oxygen consumption, metabolic rate, mean length, wet and dry weight of the exposed PL over their respective controls which can be attributed to a gradual and time-dependent accumulation of the metal, as noticed in the exposed PL through accumulation studies. Overall, the data suggest that on chronic exposure even sublethal concentrations of copper can reduce the metabolic rate and growth in P. indicus PL. This is perhaps the first attempt to use the wild P. indicus PL as a bioindicator of copper toxicity.     

Accumulation and regulation of heavy metals by the intertidal snail Polinices sordidus

The gastropod Polinices sordidus was collected from an uncontaminated area in Quibray Bay, New South Wales, Australia, in 1990. The snails were exposed for 2 wk to polluted sediments collected from Port Kembla Harbour, Blackwattle Bay, Lake Illawarra, Lake Macquarie in New South Wales, Australia, and the Derwent River in Tasmania, Australia. Metal accumulation and regulation by this species were evaluated. Metal concentrations in snail tissues and total, EDTA and HCl-extractable metals in the sediments were compared. Copper concentrations were extremely varied in snails exposed to the same sediment. This was not the case for other matals tested. No accumulation of copper was found in snails exposed to different sediments. There was no zinc accumulation from sediments containing less then 10 mg Zn/g. P. sordidus could accumulate lead, manganese and iron from some of the sediments. Manganese concentrations in the snail tissues correlated with total, HCl-extractable and, more significantly, EDTA-extractable Mn in the sediments. P. sordidus was not considered to be a good bioindicator of copper and zinc contaimination in sediments; however, this species could be used as an indicator of lead and manganese contamination.     

Immunocytochemical evidence that symbiotic algae secrete potential recognition signal molecules in hospite

Cnidarian–dinoflagellate symbioses are not well understood at the molecular level. Observed specificity between partners during initiation, establishment, and maintenance of the relationship strongly implies a role for chemical signaling. This report presents biochemical and immunocytochemical evidence for potential signaling molecules, as large molecular weight glycoproteins, secreted by Symbiodinium dinoflagellates both in culture and in symbiosis. Polyclonal antibodies directed against recovered exudate from S. microadriaticum, the natural endosymbiont of Cassiopea xamachana, the upside–down jellyfish, were highly specific in recognizing exudates from Symbiodinium species that can successfully induce developmental metamorphosis in the host but did not recognize exudates from Symbiodinium species that do not. Immunoblot analyses showed S. microadriaticum exudate to be protease sensitive. Release of antigenic material by symbiotic S. microadriaticum was demonstrated through light and electron microscopy using immunogold-labeled anti-S. microadriaticum (anti-Sm-XuLg) antibodies as probes. These secreted, symbiont-derived glycoconjugates may be candidates for interspecific molecular signals.     

Bioavailability of dissolved copper to the American oyster Crassostrea virginica. I. Importance of chemical speciation

The effect of complexation on the accumulation of dissolved copper by the American oyster Crassostrea virginica was determined in chemically defined exposure media. The speciation of copper was varied by varying the concentrations of total copper and model chelator, nitrilotriacetic acid (NTA). Accumulation of copper in 14-d experiments was related to the cupric ion activity and not the concentration of chelated copper. Rapid accumulation of copper occurred at cupric ion activities above 10^-10 M and there was no measurable accumulation at 10^-11 M.Southeast Fisheries Center Contribution No. 81-45B. Reprint request should be addressed to this author     

Mechanism of toxicity of ionic copper and copper complexes to algae

The mechanism of toxicity of ionic copper and copper complexes to growth, photosynthesis, respiration, ATP levels and mitochondrial electron-transport chain-activity in two marine diatoms, Nitzschia closterium (Ehrenberg) W. Smith (Hasle, 1964) and Asterionella glacialis Castracane, and one freshwater green alga, Chlorella pyrenoidosa Chick was investigated. Copper ions depressed both cell division and photosynthesis in A. glacialis and C. pyrenoidosa, whereas ionic copper concentrations which were inhibitory to cell division in N. closterium had no effect on photosynthesis, respiration, ATP production, electron transport or membrane ultrastructure. This suggests that in N. closterium, copper does not act on the chloroplast, the mitochondrion, or the cell membrane, since if it did, the above parameters should be affected. Copper-ethylxanthogenate was exceptional amongst the copper complexes in that it stimulated respiration, mitochondrial electrontransport and ATP formation in N. closterium under conditions of strongly inhibited cell division and slightly stimulated photosynthesis. Ionic copper toxicity may result from an intracellular reaction between copper and reduced glutathione (GSH), leading to a lowering of the GSH:GSSG ratio and suppression of mitosis. In addition, copper inhibits the enzyme catalase and reduces cell defence mechanisms against H₂O₂ and oxygen-free radicals. Lipid-soluble copper complexes are more toxic than ionic copper because both the metal and the ligand are introduced into the cell. Toxicity of ionic copper is ameliorated by trivalent metal ions in the growth medium, including those of Mn, Co, Al, Fe and Cr which form a layer of metal (III) hydroxide around the algal cell, adsorb copper and reduce its penetration into the cell. The degree of insolubility of the metal (III) hydroxide is related to its ability to protect against copper toxicity. In addition, manganese and cobalt catalytically scavenge damaging H₂O₂ and superoxide radicals, respectively, produced by the cell.     

High molecular weight algal substances in the sea

Phytoplankton exudates into culture media and extracts from littoral algae are shown to contain significant quantities of extracellular materials having molecular weights in excess of 50,000. A recent development in experimental fluid mechanics (the reduction of turbulent friction coefficients of flowing solutions by dissolved macromolecules) has been utilized as a specific test for high molecular weight compounds. Phytoplankton species in all major algal groups could be found which exude high molecular weight compounds into their culture media; the red alga Porphyridium cruentum typically produced friction reductions as high as 60%. Seaweed samples in the Chlorophyta, Phaeophyta, and Rhodophyta showed major changes in turbulent friction coefficient; in some cases the friction was less than 1/2 that of pure seawater. All samples of the genus Porphyra and Gigartina produced substantial friction changes. From these experiments it is concluded that algal exudates can be a prominent source of high molecular weight compounds in the sea.     

Inhibitory effects of copper on marine dinoflagellates

The effect of copper on three species of marine dinoflagellates [Scrippsiella faeroense (Paulsen) Balech et Soares, Prorocentrum micans Ehrenberg, Gymnodinium splendens Lebour] was studied. It inhibited the growth of all species and was lethal to one species in batch cultures. The effect was more pronounced in semicontinuous culture than in batch cultures. Chlorophyll concentrations and rate of uptake of radioactive carbon by cells of S. faeroense were affected in a manner similar to cell numbers. Copper inhibited growth of cells, most probably either by arresting cell division or by penetrating inside the cell and affecting metabolism.     

Binding of manganese by Antarctic Phaeocystis pouchetii and the role of bacteria in its release

The growth of Antarctic Phaeocystis pouchetii and associated bacteria in culture, and the binding and release of manganese were investigated using cultures derived from three clonal isolates collected from Prydz Bay, Antarctica, in November and December 1982. The cultured strains accumulated manganese from the culture medium. The concentration of Mn²⁺ in the alga was up to 58 times that of the medium. The Mn²⁺ apparently binds to the mucilage secreted by the cells and gives the alga its characteristic brown colour. Oxidation or complexing of Mn²⁺ by P. pouchetii was also indicated. Bacterial growth on the mucilage is inhibited in rapidly growing cultures by acrylic acid produced in the mucilage. Once the growth of P. pouchetii and production of ccrylic acid slow, bacterial numbers increase, leading to the solubilization of the mucilage and the release of Mn²⁺. In cool-temperate, subpolar and polar seas where P. pouchetii may form dense blooms, the binding and subsequent release of manganese may have important consequences for competing phytoplankton.     

Combined bioremediation for lead in mine tailings by Solanum nigrum L. and indigenous fungi

Lead-contaminated mine tailings were bioremediated using microbial/phyto remediation. The optimum lead accumulation and tolerance capacity of the plant–microbe partnership were investigated, and their mechanisms were evaluated further under varied levels of lead contamination through a flowerpot experiment in a greenhouse. Enzymes activities revealed that bioremediation has improved fertility and metabolism of tailing soil. The removal efficiency of lead was in the order of microbial/phytoremediation?>?phytoremediation. Solanum nigrum L. was not shown to be a hyperaccumulator for lead. Mucor circinelloides significantly enhanced the growth response and lead accumulation in plants more than Mortierella and Trichoderma asperellum. Moreover, Mortierella was discovered to have good metal tolerance capacity under high Pb concentrations (1200 and 1600?mg?kg^?1). The results for lead bioavailability showed that phytostabilisation serves as a major repair pathway for S. nigrum L. Effective fractions were immobilised for decreased bioavailability by T. asperellum and M. circinelloides. On the contrary, an increased amount of lead was mobilised for increased bioavailability by Mortierella. This study provides new insights into the feasibility of using S. nigrum L. and the aforementioned indigenous fungus strains for large-scale bioremediation of mine tailings.     

Isolation and identification of fungi tolerant to polycyclic aromatic hydrocarbons and coal tar from different habitats in Lithuania

A group of 36 fungal strains, belonging to the Lithuanian mycobiota, was collected and isolated from different locations, habitats, and matrices, including creosote-treated wood in storage yards for crosstie wastes. The eight most perspective strains selected according to preliminary assessment of tolerance to coal tar were subsequently identified combining taxonomical evaluation and molecular techniques. The tolerance of the eight identified fungal species (five basidiomycetes and three ascomycetes) to the presence of various concentrations of coal tar, and for the four most perspective fungal strains (Pleurotus sp., Schizophyllum sp., Irpex lacteus, Bjerkandera adusta) to polycyclic aromatic hydrocarbons was evaluated. The ligninolytic enzymatic activity assay of the isolated strains resulted in a good correspondence between the tolerance to pollutants and the capability to produce ligninolytic enzymes indicating that this group of white-rot fungi is perspective for further investigation and eventual usage for mycoremediation of polycyclic aromatic hydrocarbons polluted substrates.     

Differential tolerance to metals among populations of the introduced bryozoan Bugula neritina

Resistance to heavy metals is a potentially important trait for introduced marine organisms, facilitating their successful invasion into disturbed natural communities. We conducted laboratory and field experiments to examine differential resistance to copper (Cu) between two source populations of the introduced bryozoan Bugula neritina, originating from a polluted (Port Kembla Harbour, NSW, Australia) and an unpolluted (Botany Bay, NSW, Australia) environment. A laboratory toxicity test was conducted to test the relative resistance of B. neritina recruits from the two sources, by measuring the attachment success, survival and growth of individuals exposed to a range of Cu concentrations (0, 25, 50 and 100 μg l⁻¹ Cu). Upon completion, reciprocal transplantation of the colonies to the original polluted and unpolluted locations was carried out to assess ongoing survival and growth of colonies in the field. B. neritina colonies originating from the polluted Port Kembla Harbour had increased resistance to Cu relative to populations from an unpolluted part of Botany Bay. There appeared to be a cost associated with increased metal tolerance. In the laboratory, Botany Bay recruits displayed significantly higher growth in control treatments and significantly poorer growth at 100 μg l⁻¹ Cu with respect to Port Kembla Harbour individuals, which showed unusually uniform and low growth irrespective of Cu concentration. No difference in attachment success or post-metamorphic survival was observed between populations. Field transplantation showed copper resistance in Port Kembla Harbour colonies constituted an advantage in polluted but not benign environments. The findings of this study provide evidence of the benefits to invasive species of pollution tolerance and suggest that human disturbance can facilitate the establishment and spread of invasive species in marine systems.