COPPER‐UPTAKE KINETICS OF COASTAL AND OCEANIC DIATOMS1

We investigated copper (Cu) acquisition mechanisms and uptake kinetics of the marine diatoms Thalassiosira oceanica Hasle, an oceanic strain, and Thalassiosira pseudonana Hasle et Heimdal, a coastal strain, grown under replete and limiting iron (Fe) and Cu availabilities. The Cu‐uptake kinetics of these two diatoms followed classical Michaelis–Menten kinetics. Biphasic uptake kinetics as a function of Cu concentration were observed, suggesting the presence of both high‐ and low‐affinity Cu‐transport systems. The half‐saturation constants (K_m) and the maximum Cu‐uptake rates (V_max) of the high‐affinity Cu‐transport systems (～7–350 nM and 1.5–17 zmol · μm^?2 · h^?1, respectively) were significantly lower than those of the low‐affinity systems (>800 nM and 30–250 zmol · μm^?2 · h^?1, respectively). The two Cu‐transport systems were controlled differently by low Fe and/or Cu. The high‐affinity Cu‐transport system of both diatoms was down‐regulated under Fe limitation. Under optimal‐Fe and low‐Cu growth conditions, the K_m of the high‐affinity transport system of T. oceanica was lower (7.3 nM) than that of T. pseudonana (373 nM), indicating that T. oceanica had a better ability to acquire Cu at subsaturating concentrations. When Fe was sufficient, the low‐affinity Cu‐transport system of T. oceanica saturated at 2,000 nM Cu, while that of T. pseudonana did not saturate, indicating different Cu‐transport regulation by these two diatoms. Using CuEDTA as a model organic complex, our results also suggest that diatoms might be able to access Cu bound within organic Cu complexes.     

Effect of Copper Ions on Early Developmental Stages of the Mussel <Emphasis Type="Italic">Mytilus trossulus</Emphasis> (Bivalvia)

The effect of exposure to copper in seawater (0.005, 0.01, and 0.02 mg/liter) on the development of Mytilus trossulus was examined in the stages of fertilization, blastula, early veliger, veliger, and veliconch. Copper in a concentration of 0.01 and 0.02 mg/liter inhibited the development and growth of the embryos and larvae. At 0.005 mg Cu/liter, the embryos and larvae were capable of adaptation. If M. trossulus embryos and larvae were maintained for 1–2 days in seawater containing 0.01 mg Cu/liter and then transferred to clean water, the consequences were largely dependent on the developmental stage at which the exposure to copper took place. The early developmental stages were more sensitive to the effect of copper than veliger larvae.Original Russian Text Copyright ¢ 2005 by Biologiya Morya, Yaroslavtseva, Sergeeva.     

SELENIUM: AN ESSENTIAL ELEMENT FOR GROWTH OF THE COASTAL MARINE DIATOM THALASSIOSIRA PSEUDONANA (BACILLARIOPHYCEAE)1,2

An obligate requirement for selenium is demonstrated in axenic culture of the coastal marine diatom Thalassiosira pseudonana (clone 3H) (Hust.) Hasle and Heimdal grown in artificial seawater medium. Selenium deficiency was characterized by a reduction in growth rate and eventually by a cessation of cell division. The addition of 10⁻¹⁰ M Na₂SeO₃ to nutrient enriched artificial seawater resulted in excellent growth of T. pseudonana and only a slight inhibition of growth occurred at Na₂SeO₃ concentrations of 10⁻³ and 10⁻² M. By contrast, Na₂SeO₄ failed to support growth of T. pseudonana when supplied at concentrations less than 10⁻⁷ M and the growth rate at this concentration was only one quarter of the maximum growth rate. The addition of 10⁻³ and 10⁻² M Na₂SeO₄ to the culture medium was toxic and cell growth was completely inhibited. Eleven trace elements were tested for their ability to replace the selenium requirement by this alga find all were without effect. In selenium-deficient and selenium-starved cultures of T. pseudonana cell volume increased as much as 10-fold as a result of an increase in cell length (along the pervalvar axis) but cell width was constant. It is concluded that selenium is an indispensable element for the growth of T. pseudonana and it should be included as a nutrient enrichment to artificial seawater medium when culturing this alga.     

Effects of copper enrichment on survival,growth and photosynthetic pigment of seedlings and young plants of the eelgrass Zostera marina

Copper (Cu²⁺) is an essential nutrient for plants but toxic at high concentrations. We subjected seedlings and young plants of eelgrass Zostera marina to different seawater Cu concentrations (3, 4, 5, 10, 30 and 50?µg?l^?1) for over 30 days under controlled laboratory conditions. Natural seawater without added Cu (3?µg?l^?1) was used as reference seawater. We measured plant response in terms of survivorship, morphology, growth, productivity and leaf pigment concentration. Survival analysis combined with morphological, dynamic and productive assessment suggested that the optimum seawater Cu concentration for the establishment of Z. marina seedlings and young plants is 4?μg?l^?1. The photosynthetic response of young plants to copper enrichment, including an increase in chlorophyll content under low Cu concentration treatment but significant decrease when treated with high concentrations of Cu, is similar to those reported for other seagrass species. NOEC (no observed effect concentration), LOEC (lowest observed effect concentration) and LC₅₀ (lethal concentration that caused an increase in mortality to 50% of that of the control) values of seedlings were significantly lower than those of young plants, implying a reduced Cu tolerance to high concentrations (>10?μg?l^?1). This study provides data that could prove helpful in the development of successful eelgrass restoration and conservation.     

Copper biosorption by Pseudomonas cepacia and other strains

Biosorption of copper by Pseudomonas cepacia was found to be dependent on added copper concentration. Copper uptake by the cells was rapid over the range of copper concentrations tested and complete within the first 10 min of incubation time. The effect of pH on copper uptake by P. cepacia was determined using overlapping buffers over the pH range 3–8, and copper biosorption from a 10 mM copper solution was greatest at pH 7. Copper uptake (measured by analysis of cell digests) was unaffected by cyanide and azide (up to 30 mM) and by incubation of cells with a 10 mM copper solution at 4 °C. Evidence from these results suggested that copper uptake by P. cepacia cells involves surface binding and not intracellular accumulation by active transport. Biosorption of copper by various Pseudomonas isolates from metal-contaminated environments agreed well with copper biosorption by Pseudomonas strains from the National Collection of Type Cultures (NCTC).     

Effects of high copper concentrations on soil invertebrates (earthworms and oribatid mites):

Data in the literature on the toxicity and uptake of copper by soil invertebrates are contradictory. Copper toxicity and bioaccumulation studies were therefore performed using earthworms and oribatid mites. Field-simulating experiments in soil-filled plastic containers showed that earthworms try to escape moderately toxic situations and that they are much more sensitive than oribatid mites to temporary high Cu²⁺ concentrations in soils. The total copper concentration in the bodies of the earthworm species Octolasium cyaneum was measured in experiments with different soil types and different amounts of added CuSO₄. The copper concentrations in the earthworms increased in response to the higher concentrations of the copper fraction extractable with 2.5% acetic acid in the soil. Furthermore, internal copper concentrations showed a slight tendency to oscillate. The worms died when the concentrations within their bodies exceeded about 100–120 ppm, calculated on a dry weight basis. To interpret the experimental results, a compartment model is proposed which describes the dynamics of different fractions of copper in worms living in varying soil environments. Applying this model, the different reports on toxicity and uptake of copper in the literature no longer contradict each other.     

UPTAKE AND RELEASE OF METHYLMERCURY-203 BY CLADOPHORA GLOMERATA1

Cladophora glomerata was exposed to CH₃²⁰³HgCl at concentrations of 10, 50, and 100 μg/liter of water. Formalin-killed alga was exposed to a concentration of 50 μg CH₃²⁰³HgCl/liter. Uptake was monitored at 2 and 12 hr, and days 1, 2, 4, 8, and 16. At the end of this period, the Cladophora was placed in uncontaminated water, and release of methylmercury was monitored at 1, 2, 4, 8, and 16 days. Sorption occurred at all concentrations, and the live algal material accumulated more methylmercury than the dead alga, at equal exposure concentrations. Accumulation of methylmercury by the live Cladophora peaked on or near the second day for all exposure concentrations, suggesting that the uptake rate was independent of methylmercury concentration in the water. Uptake was greatest at the 50 μg/liter exposure. Desorption was nominal during the 16-day release period. The mechanisms of methylmercury uptake by Cladophora are discussed.     

EFFECT OF COPPER ON ALGAL COMMUNITIES FROM OLIGOTROPHIC CALCAREOUS STREAMS1

Two sets of experiments were done to quantify the effects of chronic copper exposure on natural peri‐ phyton in a nonpolluted calcareous river. The results of short‐term (up to 6 h exposure) experiments corroborated the significance of pH on copper toxicity. Copper toxicity increased when pH was reduced from 8.6 to 7.7, and this was related to the effect of pH on copper speciation (free copper concentration increased from 0.2% to 2.3% of total copper). Longer term experiments demonstrated that periphyton communities exposed to copper under pH variation (8.2–8.6) were already affected at 10 μg·L ^{? 1} (20–80 ng·L ^{? 1} Cu^{2 +} ) after 12 days of exposure. Copper exposure caused stronger effects on structural (algal biomass and community structure) than on functional (photosynthetic efficiency) parameters of peri‐ phyton. Changes in community composition included the enhancement of some taxa (Gomphonema gracile), the inhibition of others (Fragilaria capucina and Phormidium sp.), and the appearance of filament malformations (Mougeotia sp.). The results of our study demonstrated that several weeks of exposure to copper (10–20 μg·L ^{? 1}) were sufficient to cause chronic changes in the periphyton of oligotrophic calcareous rivers. This degree of copper pollution can be commonly found in the Mediterranean region as a result of agricultural practices and farming activities.     

Population responses of the freshwater amphipod crustacean Gammarus pulex (L.) to copper

1. Populations of Gammarus pulex (density 4000m^?2) were exposed, over a 100-day period in a continuous-flow dosing system, to concentrations of copper below the 240 h median lethal concentration (LC₅₀) for juveniles. 2. There was a significant effect of copper on the resulting population density. In the control and lowest treatment (11.0μg1-^?1) it was double the initial density, but with increasing copper concentration there was a decrease in density until at the highest treatment (23.1 μg1-^?1), it was less than that of the initial population. The lowest observed effect concentration (LOEC) for population density was 14.6 μg 1-^?1. 3. Copper significantly affected the age composition of the final populations. In the control and lowest treatment concentration, the population was composed mainly of juvenile animals. With increasing copper concentration, however, there was a decrease in the number of juveniles, the LOEC being 14.6 μg1-^?1. The number of adults in the final population was also significantly reduced by copper, with an LOEC of 18.2 μg1-^?1. 4. The relationship between the body length and number of antennal segments of animals in the populations at each treatment concentration showed that growth was impaired by increased copper concentrations.     

Effects of copper and other metals on fertilization,embryo development,larval survival and settlement of the polychaete Nereis (Neanthes) virens

Summary

Nectochaete larvae of the ecologically and economically important ragworm, Nereis virens, were exposed to cadmium, chromium, copper, lead and zinc dissolved in seawater to nominal concentrations ranging from 0 to 5000 μg l^?1. Copper was the most toxic (mean LC50 of 76.5 μg l^?1 ± 95% CI 73.8–79.2 after 96 h exposure) and so was used for subsequent experiments. Exposure of gametes to greater than 500 μg l^?1 copper for 2 or 4 h at 10°C prior to fertilization, or a 10 min exposure during fertilization, significantly reduced embryo developmental success. The effect of copper on larval settlement was also assessed using sediment spiked to a range of concentrations (0, 50, 250, 500, 1000 mg kg!1 dry weight). Significantly fewer larvae were found in sediment of $250 mg kg!1 in comparison to the control or the 50 mg kg!1 treatment. Assessment of living larvae also confirmed a significant reduction in settlement, but in all treatments compared to the control, although the number of dead larvae also increased as the concentrations increased. These effects may have important implications for reproductive success and recruitment of N. virens to polluted sediments.     

Accumulation,regulation and toxicity of copper,zinc, lead and mercury in Hyalella azteca

Zinc, lead and mercury accumulation in the amphipod Hyalella azteca increases with increasing exposure to metals. During 10 week chronic toxicity tests, metal accumulated at the highest non-toxic/lowest toxic concentration was 126/136 µg Zn g^–1, 7.1/16 µg Pb g^–1 and 56/90 µg Hg g^–1 dry weight. Concentrations of lead and mercyry in control animals were substantially lower (1.3 µg Pb g^–1 and 0.4 µg Hg g^–1), but concentrations of zinc in controls (74 µg g^–1) were about one half those of the lowest toxic concentration. Copper was completely regulated. Accumulated copper concentrations after 10 weeks exposure to all waterborne copper concentrations resulting in less than 100% mortality were not significantly different from controls (79 µg g^–1). Lead and mercury concentrations in wild H. azteca should be useful indicators of potential toxicity. Zinc accumulation may also be a useful indicator of zinc toxicity, but careful comparison with control or reference animals is necessary because of the small differences between toxic and control concentrations. Copper is not accumulated by H. azteca under chronic exposure conditions and body burdens of field animals cannot be used as an indicator of exposure or potential toxic effects. Short term exposures to copper, however, result in elevated copper concentrations in H. azteca, even at concentrations below those causing chronic toxicity. Short term bioaccumulation studies might, therefore, provide a useful indication of potential chronic copper toxicity.     

COPPER TOXICITY TO SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1,2

Copper toxicity to Skeletonema costatum (Grev.) Cleve has been studied in batch cultures of chemically defined culture media. The alga is relatively insensitive to cupric ion activity, demonstrating no effect on growth up to (Cu²⁺) = 10^?8.5 M. Cultures inoculated from stationary phase stocks exhibit a prolongation of the lag phase with increasing copper concentrations near and above the point of precipitation of the copper. The toxicity of copper is a function of the silicic acid concentration in the medium. This effect is observed in a range of Si(OH)₄ concentrations (10^?5 M to 10^?4 M) above known values for the saturation of silicon uptake kinetics, thus suggesting an influence of copper on silicate metabolism.     

THE UPTAKE OF GLUCOSE BY CHLAMYDOMONAS SP.12

The glucose uptake of a species of Chlamydomonas was studied at various concentrations of d -glucose plus glucose-1-¹⁴C (0.003–10.0 mg/liter) and at various light levels (0–220 ft-c). The alga grows at 4 C either in the light or in the dark with added glucose, cellobiose, maltose, or fructose. Uptake of glucose could be described by the Michaelis-Menten equation, and both the maximum velocity of uptake and the half-saturation constant increased when the cells were exposed to glucose in the dark. However, the high value of the half-saturation constant (5 mg glucose/liter) compared with the low levels of glucose in nature (5–10 μg/liter) makes it unlikely that a transport system is effective under natural conditions. Even if a total of 10.0 mg/liter of glucose plus other organic compounds were available as substrate, the rate of photosynthesis would still be more than 10 times higher (at 220 ft-c) than the rate of organic substrate uptake. Light had no effect on the total uptake of glucose but did reduce the percentage of ¹⁴CO₂ evolved from 61% of the total ¹⁴CO taken up in the dark to 0% at 220 ft-c. This decrease could be due to either preferential use of the ¹⁴CO₂ in photosynthesis or of the photosynthate in respiration.     

EFFECT OF THE SINKING RATE OF TWO DIATOMS (THALASSIOSIRA SPP.) ON UPTAKE FROM LOW CONCENTRATIONS OF PHOSPHATE1

The effect of the sinking rate, or rate of medium flow (φ) on the rate of phosphate incorporation (V) by the planktonic diatoms Thalassiosira fluviatilis Hust. and T. pseudonana Hasle & Heimdal in batch and chemostat cultures was determined by passing medium at defined flow rates (0.5–25.0 mm·min^?1) over algae on membrane filters. At concentrations from 1 to 100 μg phosphorus·l^?1 V, increases with increasing velocity of flow, approaching a maximum value (V_m) as described by the empirical relationship: where K_φ is the sinking rate value when V = 1/2 V_m+ V_o and V_o is the uptake at 0 rate of flow. By comparing uptake at controlled flow with uptake in a vigorously stirred medium, the phosphate concentration in the cell boundary layer can be determined. The sinking rate that reduces the phosphate concentration in the boundary layer to half of nominal concentration in the medium is much lower for the larger T. fluviatilis than for T. pseudonana. For both diatoms, it is inversely related to the nominal concentration.     

Effects of PCB on interspecific competition in natural and gnotobiotic phytoplankton communities in continuous and batch cultures

The toxicity of polychlorinated biphenyls (PCB) to the diatomThalassiosira pseudonana (formerlyCyclotella nana), grown in pure and mixed cultures, was greatest when in competition with other species. Continuous cultures were superior to batch cultures for studying competitive interactions, and PCB caused greater alteration of species composition in continuous cultures than it did in batch cultures. Natural phytoplankton communities from Vineyard Sound, maintained in continuous culture, responded to PCB stress the same as did gnotobiotic communities, withT. pseudonana showing similar responses in both communities. A PCB concentration of 0.1 μg/liter (0.1 part per billion), a level not uncommon in natural waters, did not affect algal growth in pure cultures but caused substantial disruption of continuous culture communities. The possible impact of PCB pollution on natural phytoplankton communities is discussed. Contribution No. 3181 from the Woods Hole Oceanographic Institution.     

Copper uptake inAspergillus niger during batch growth and in nongrowing mycelial suspensions

Copper accumulation by the filamentous fungusAspergillus niger from a glucose mineral salts medium containing copper in the concentration range 16 to 157 μM was maximal in the lag phase of growth. In the subsequent linear growth phase, the mycelial copper contents were dramatically reduced on a per gram dry weight basis. The fungal mycelium exhibited pelleted morphology and exponential growth was not apparent. The medium pH was reduced during growth in flask cultures, but this was not responsible for the reduction in copper uptake as indicated by the similar effect in cultures grown in a stirred-tank fermenter with electronic maintenance of pH at 5.5. Voltammetric analysis of medium which had supported growth of the fungus showed that copper added at a final concentration of 40 μM was complexed. Energy-dependent copper uptake from 2-(N-morpholino)ethanesulfonic acid buffer at pH 5.5 containing 40 μM copper could not be demonstrated in nongrowing mycelium. Incubation at 4°C reduced copper uptake while the presence of 10 mM glucose or preincubation of the mycelium in 1 mM sodium azide had no effect on copper uptake.     

Life-history traits of a tube-dwelling corophioid amphipod, Paracorophium excavatum, exposed to sediment copper

This is the first demonstration that sediment contaminants can influence the reproduction of amphipods. Groups of Paracorophium excavatum from a slightly contaminated estuarine site were held within laboratory mesocosms containing four copper-spiked estuarine sediments (Cu 14-46 μg g⁻¹ dry weight) and a control sediment (Cu 5 μg g⁻¹ dry weight) at 15 °C for 28 days. Copper sediment concentration did not affect the amphipod sex ratio. Female maturation was inhibited within copper-spiked sediments but female length was similar. Juvenile recruitment occurred only in sediments containing less than 20 μg g⁻¹. Males were significantly larger than females in the control sediment (Cu 5 μg g⁻¹ dry weight) and male length decreased linearly with increasing copper concentration. The copper concentration within whole body tissues increased with dry body dry weight in all sediments except the highest copper concentration. Following 28 days of exposure, none of the female amphipods from the copper-dosed sediments was brooding embryos. In contrast, brood size of females in the control sediment (Cu 5 μg g⁻¹) was similar to field samples. Because low concentrations of sediment copper affect the maturation and growth rates of male and female amphipods differently, these life-history traits could affect the population structure of amphipods exposed to copper contaminated sediments.     

Metal uptake by different species of phytoplankton in culture

Cadmium and copper uptake by cultures of Prasinocladus marinus, Amphidinium carterae, Chaetoceros curvisetum and Chaetoceros protuberans was considered as a function of exposure time (from 0.5 to 72 h) and of metal concentrations added to the culture medium (from 5 to 20 µg l^–1). For cadmium uptake, A. carterae exhibited a particular behaviour: the accumulation was not dependent on exposure time or on metal concentration. An adsorption process appeared to govern both Cadmium uptake by phytoplankton species (except A. carterae) and that of copper. In addition to adsorption, another active process might be involved in the Cadmium uptake of P. marinus, C. curvisetum and C. protuberans.     

Assessment of heavy metal accumulation in Anzali wetland,Iran, using a submerged aquatic plant,Ceratophyllum demersum

The objective of this study in 2009 was to examine whether levels of cadmium (Cd), copper (Cu), zinc (Zn), lead (Pb) and chromium (Cr) were higher in the leaves than in the stems of a submerged aquatic plant Ceratophyllum demersum in Anzali wetland. Cadmium, Pb and Cr concentrations were highest in the leaves. The mean concentrations of Cd and Cr in the leaves at all the sampling sites ranged between 0.94–1.26 μg g^?1 and 1.03–2.71 μg g^?1, respectively. Lead also had its highest concentrations in the leaves. The mean concentration of Pb in the leaves at all sampling sites ranged between 7.49–11.88 μg g^?1. Copper and Zn concentrations were highest in the stems. The mean concentrations of Cu and Zn in the stems at all sampling sites ranged between 10.79–17.91 μg g^?1 and 19.89–40.01 μg g^?1, respectively. Cadmium and Pb concentrations were higher in the leaves than in the stems, while Zn concentration was higher in the stems than in the leaves. Accumulation of Cu and Cr in the organs of C. demersum was in descending order of leaf ～ stem, since there was no significant difference between their mean concentrations in the leaves and stems.     

Possible mechanisms underlying copper-induced damage in biological membranes leading to cellular toxicity

It is generally accepted that copper toxicity is a consequence of the generation of reactive oxygen species (ROS) by copper ions via Fenton or Haber-Weiss reactions. Copper ions display high affinity for thiol and amino groups occurring in proteins. Thus, specialized proteins containing clusters of these groups transport and store copper ions, hampering their potential toxicity. This mechanism, however, may be overwhelmed under copper overloading conditions, in which copper ions may bind to thiol groups occurring in proteins non-related to copper metabolism. In this study, we propose that indiscriminate copper binding may lead to damaging consequences to protein structure, modifying their biological functions. Therefore, we treated liver subcellular membrane fractions, including microsomes, with Cu²⁺ ions either alone or in the presence of ascorbate (Cu²⁺/ascorbate); we then assayed both copper-binding to membranes, and microsomal cytochrome P450 oxidative system and GSH-transferase activities. All assayed sub-cellular membrane fractions treated with Cu²⁺ alone displayed Cu²⁺-binding, which was significantly increased in the presence of Zn²⁺, Hg²⁺, Cd²⁺, Ag⁺¹ and As³⁺. Treatment of microsomes with Cu²⁺ in the μM range decreased the microsomal thiol content; in the presence of ascorbate, Cu²⁺ added in the nM concentrations range induced a significant microsomal lipoperoxidation; noteworthy, increasing Cu²⁺ concentration to ≥50 μM led to non-detectable lipoperoxidation levels. On the other hand, μM Cu²⁺ led to the inhibition of the enzymatic activities tested to the same extent in either presence or absence of ascorbate. We discuss the possible significance of indiscriminate copper binding to thiol proteins as a possible mechanism underlying copper-induced toxicity.