Marine Diatom Nitzschia Closterium Research Articles

Optimal chlorophyll fluorescence parameter selection for rapid and sensitive detection of lead toxicity to marine microalgae Nitzschia closterium based on chlorophyll fluorescence technology

Heavy metal pollution as one of the most serious pollution problems of marine environment, seriously threatens the safety of marine organism and human health, and will lead to potential risks for the marine ecological environment. In order to develop a rapid and sensitive toxicity detection method for marine heavy metals, in this study, marine diatom Nitzschia closterium was used as the test organism, and the effects of different concentrations of lead (Pb) on the five chlorophyll fluorescence parameters of N. closterium including the maximum photochemical quantum yield of PSII (Fv/Fm), the effective quantum yield of PSII photochemical energy conversion (ΦPSII), the effective absorption cross section of PSII photochemistry (σPSII′), the relative electron transfer rate of PSII (rP), and the PSII electron flux per unit volume (JVPII) at different exposure times were investigated based on chlorophyll fluorescence technology. By comparing with the photosynthetic activity fluorescence parameter Fv/Fm which is commonly used for toxicity analysis of pollutants using algae as test organisms, the optimal chlorophyll fluorescence parameter that could rapidly and sensitively determine Pb toxicity to N. closterium was selected. The results indicate that all the five chlorophyll fluorescence parameters of Fv/Fm, ΦPSII, σPSII′, rP and JVPII showed good dose-response relationships with Pb within 8 h exposure time, and they all could be used as endpoints to rapidly determine Pb toxicity to N. closterium. Among the five chlorophyll fluorescence parameters, JVPII was the most sensitive fluorescence parameter for detecting the toxicity of Pb to N. closterium within 6 h exposure. And for JVPII, the median effective concentration (EC50) values of Pb at 2, 4 and 6 h were 0.329, 0.068 and 0.040 mmol L−1, respectively. However, when the exposure time was 8 h, ΦPSII was the most sensitive fluorescence parameter for the toxicity detection of Pb, and the EC50 value of Pb at 8 h was 0.038 mmol L−1. This study will provide an important basis for the development of a rapid and sensitive detection method for the biological toxicity of marine heavy metals, and those results will be helpful for ecological risk assessment in marine environment.

Silicon limitation reduced the adsorption of cadmium in marine diatoms

The nitrogen (N) and phosphorus (P) loadings in coastal waters have greatly increased due to anthropogenic input. However, the silicate levels have remained stable in the past decades, leading to nutrient status alternating from N limitation to Si limitation. Few studies have examined the effects of such nutrient imbalance on Cd accumulation in marine diatoms. In the present study, we provide multiple lines of evidence that Si limitation reduced the Cd binding capability in the marine diatom Nitzschia closterium. Diatom cells adapted to lower Si exposure had weaker mechanical strength and less negatively charged surfaces. Close examination of the cell surface revealed that Si shortage changed the biochemical composition of the cell surface and decreased the diatom’s silicification ability. The lower density of the silanol groups in the cell wall is the most likely reason for the weaker Cd adsorption ability observed in the Si limited diatom cells. This study demonstrates the significance of Si in controlling the metal accumulation in marine diatoms.

The effect of bacteria on the sensitivity of microalgae to copper in laboratory bioassays

Although single-species laboratory toxicity tests with microalgae are sensitive and highly reproducible, they lack environmental realism. Interactions between algae and their associated bacteria, either in the plankton or in biofilms, may alter algal sensitivity to contaminants, which are not mimicked in laboratory toxicity tests. This study investigated the effects of simple algal-bacterial relationships on the sensitivity of laboratory-cultured algae to copper using 72-h algal growth-rate inhibition bioassays. Four species of microalgae were used, two isolates of each; a strain of algae with no microscopically visible and no culturable bacteria present (operationally defined as axenic) and a non-axenic strain. The four algae used were the marine diatom Nitzschia closterium, the freshwater green alga Pseudokirchneriella subcapitata and two tropical Chlorella spp. Under control conditions (no copper), N. closterium and P. subcapitata grew better in the presence of the bacterial community. Sensitivity to copper (assessed as the concentration to inhibit the growth rate by 50% after 72-h (IC50)) was not significantly different for the axenic and non-axenic strains of N. closterium, P. subcapitata or for Chlorella sp. (PNG isolate). At pH 5.7, the axenic Chlorella sp. (NT isolate) had a 72-h IC50 of 46 μg Cu L −1, while in the presence of bacteria the IC50 increased (i.e., sensitivity decreased) to 208 μg Cu L −1. However, when the bacterial status of both the operationally defined axenic and non-axenic cultures of N. closterium and Chlorella sp. (NT isolate) was investigated using polymerase chain reaction (PCR) amplification of 16S rRNA followed by DNA fingerprinting using denaturing gradient gel electrophoresis (DGGE), it was found that bacteria were actually present in all the algal cultures, i.e. the axenic cultures were not truly bacteria-free. Based on sequence information, the bacteria present were nearly all identified as alphaproteobacteria, and a number of isolates had high similarity to bacteria previously identified as symbionts or species endophytically associated with marine organisms. The “axenic” cultures contained less bacterial phylotypes than the non-axenic cultures, and based on band-intensity, also contained less bacterial DNA. This supported the findings of few differences in copper sensitivity between strains, and suggests that standard microalgal toxicity tests probably inadvertently use non-axenic cultures in metal assessment.

Copper and zinc tolerance of two tropical microalgae after copper acclimation

Current toxicity tests with microalgae are often criticized as being overly sensitive to metals because algae are cultured in metal-deficient media. If such bioassays overestimate copper toxicity in surface waters, the relevance of water quality guidelines derived from these tests is questionable. In this study, the effect of acclimation to copper at environmentally relevant concentrations, on the sensitivity of the marine diatom Nitzschia closterium and the freshwater green alga Chlorella sp. to copper and zinc was examined. N. closterium was acclimated in culture medium containing 5 or 25 microg Cu L(-1) for 200 days, while Chlorella sp. was acclimated in medium containing 2 microg Cu L(-1) for 100 days. Changes in algal growth rates and copper and zinc tolerance were monitored using standard growth inhibition toxicity tests in minimal medium over 72 h. Neither of the two acclimated N. closterium cultures had increased zinc or copper tolerance compared with that of the nonacclimated algae, nor were there any changes in control growth rates. Similarly, no changes in copper tolerance or control growth rates were observed for the acclimated Chlorella sp. culture. This was supported by measurements of intracellular and extracellular copper which confirmed that there were no differences in copper accumulation in either acclimated or nonacclimated algae. These results suggest that these algae grown in standard culture media are generally no more sensitive than algae grown in a metal-enriched medium. This supports the continued use of current laboratory bioassays with microalgae, as part of a suite of tests for assessing metal bioavailability, for use in ecological risk assessments and for providing data for the derivation of water quality guidelines.

The Development of Marine Toxicity Identification Evaluation (TIE) Procedures Using the Unicellular Alga Nitzschia closterium

Unicellular algae are highly sensitive to a wide range of toxicants and have been used extensively in ecotoxicological testing. This, along with their ability to grow in very small test volumes over short test durations, make them ideal test organisms for use in Toxicity Identification Evaluations (TIEs). Despite this, microalgae have not previously been used in marine TIE studies. In this study, the marine diatom Nitzschia closterium was shown to be a highly suitable test organism after modification of the standard test protocol to reduce test volumes to 6 mL and test duration to 48 h. The alga was tolerant to the chemicals used in phase I of the standard USEPA TIE protocol, and physical TIE manipulations had no effect on algal growth. The cation exchange procedure, however, inhibited algal growth, while the anion exchange procedure stimulated growth, making these two procedures unsuitable for use with this species. Of the buffers trialed for the graduated pH procedure, 0.01 M PIPES buffer was found to be suitable for buffering at pH 7 because it maintained the required pH over the duration of the test and did not affect the growth or sensitivity of the algae to one reference toxicant (copper). A trial TIE on a secondary-treated sewage effluent for discharge into coastal waters showed that the developed protocols could successfully be used to identify ammonia as the major toxicant in the effluent.

Colonization and community dynamics of class Flavobacteria on diatom detritus in experimental mesocosms based on Southern Ocean seawater

In order to better understand the ecology of microorganisms responsible for secondary production in the Southern Ocean the activity of Flavobacteria communities on diatom detritus in seawater mesocosms was investigated. Seawater was collected from different parts of the Southern Ocean including the Polar Front Zone (PFZ), ice-edge area of the Antarctic Zone (AZ), and a site in the AZ ice pack. Detritus from the cosmopolitan marine diatom Nitzschia closterium Ehrenberg was resuspended in mesocosms containing seawater filtered to remove particulate organic matter, including particle-associated bacteria and most eukaryotes, but retaining native planktonic bacterial assemblages. Mesocosms were incubated at 2 °C and samples analysed for changes in community composition using denaturing gradient gel electrophoresis (DGGE), real-time PCR and fluorescent in-situ hybridization (FISH). DGGE banding patterns and FISH images demonstrated rapid bacterial colonization of the detritus, dominated by members of class γ- Proteobacteria, α- Proteobacteria and Flavobacteria. Real-time PCR data indicated members of class Flavobacteria were involved in initial colonization of detrital aggregate, however relative abundance stayed at similar levels found for the original native particle-associated populations. 16S rRNA gene DGGE banding patterns and sequence analysis demonstrated significant variation in Flavobacteria community structure occurred in the first 20 days of the experiment before community stabilization occurred. The community structures between the three mesocosms also markedly differed and major colonizers were primarily derived from detectable members of the initial particle-associated Flavobacteria community, however the abundant uncultured Flavobacteria agg58 clone-related and DE cluster 2 clades, previously identified in Southern Ocean seawater were not observed to colonize the detritus.

Toxicity of cyanide and cyanide complexes to the marine diatom Nitzschia closterium

The effect of sodium cyanide (NaCN), iron-cyanide complexes and a blast furnace effluent on the growth of the unicellular marine diatom Nitzschia closterium was investigated. Due to the volatile nature of HCN, the free and total cyanide concentrations in all test solutions were monitored daily to ensure that average daily losses of free cyanide did not exceed 5%. The iron-cyanide complexes were less toxic than the NaCN, with 72-h EC 50 values of 57, 127 and 275 μg total-CN litre −1 for NaCN, K 3Fe(CN) 6 and K 4Fe(CN) 6, respectively. The estimated lowest observed effect concentration (LOEC) for NaCN was 10 μg litre −1. The toxicity of the cyanide complexes was largely due to free cyanide (HCN + CN −) with possible additional toxicity caused by dissociation of the complexes at the algal cell membrane. The toxicity of an effluent from a blast furnace drawdown episode also correlated well with the toxicity of free cyanide.

Toxicity of lipid-soluble copper(II) complexes to the marine diatom Nitzschia closterium: Amelioration by humic substances

Algal assays, using the marine diatom Nitzschia closterium, have established that humic acid (5 mg kg −1) can ameliorate the toxicity of the lipophilic complex Cu(oxine) 2 (3 × 10 −8 mol l −1 in unsupplemented seawater). The toxicity of Cu(PAN) 2 is not ameliorated [PAN = 1-(2-pyridylazo)-2-napthol]. In conjunction with previous visible absorption spectrophotometry and polarographic measurements it was established that humic acid sequesters copper(II) from the hydrophobic complexes, releasing a ligand molecule. The copper(II) toxicity may be ameliorated provided the ligand itself is not toxic. Fulvic acid was significantly less effective in ameliorating toxicity. Because of the significant competition from Ca(II) and Mg(II) in seawater, it is inferred that humic substances may be more effective in ameliorating toxicity of hydrophobic copper complexes in fresh water.

Mechanism of toxicity of zinc to the marine diatomNitzschia closterium

The effect of zinc on cell division, photosynthesis, ultrastructure, respiration, ATP levels, mitochondrial electron-transport chain (ETC)-activity, total thiols and glutathione in the marine diatomNitzschia closterium (Ehrenberg) W. Smith was investigated. Although 65µg Zn 1−1 halved the cell division rate, photosynthesis and respiration were unaffected by zinc concentrations up to 500µg Zn 1−1. Most of the zinc associated with the cells was bound at the cell surface, with only 3 to 4% of this extracellular zinc penetrating the cell membrane. Once inside the cell, zinc exerted its toxicity at a number of sites. Increased ATP production and ETC activity were observed in zinc-treated cells. Zinc also enhanced cellular thiols (SH) and total glutathione, and zinc toxicity was reversible by the addition of thiol compounds such as cysteine. Zinc-thiol binding may be a detoxification mechanism for the cell. It is suggested that increased ATP production may provide the energy required for increased glutathione synthesis at the expense of other energy-requiring processes including cell division. The mechanisms of toxicity of ionic zinc and copper toN. closterium were compared.

The effect of culture medium on metal toxicity to the marine diatom Nitzschia closterium and the freshwater green alga Chlorella pyrenoidosa

The composition of the culture medium and its effects on the toxicity of ionic copper and zinc to the marine diatom Nitszchia closterium and the toxicity of copper and lead to the freshwater green alga Chlorella pyrenoidosa were examined. The inhibitory concentration of copper giving 50% reduction in cell division rate in C. pyrenoidosa increased from 16 μg Cu l −1 in synthetic softwater and 24 μg Cu l −1 in EPA-AAP medium (minus EDTA) to >200 μg Cu l −1 in high nutrient MBL medium. Similarly, marine medium f reduced copper and zinc toxicity to N. closterium 10–20 fold compared to seawater. To overcome the problems of metal complexation by chelators such as silicate, iron and EDTA in the assay medium, unsupplemented seawater and buffered synthetic softwater, enriched only with nitrate and phosphate, have been used for the study of metal toxicities to marine and freshwater algae. These low nutrient media are sufficient to support algal growth over the 4-day assay period.

Manganese catalysis of dopamine oxidation

Manganese catalysis of the oxidation of dopamine by air was studied as part of an investigation of possible manganese intoxication amongst a group of Aborigines living on manganese-rich soil on Groote Eylandt, in the Northern Territory of Australia. Manganese significantly increased the oxidation rate of dopamine, and the manganese complexes with some purines were especially efficient catalysts. An oxidation mechanism, involving a manganese(II)/(III) redox couple and a semiquinone free radical intermediate, is proposed. Stoichiometric hydrogen peroxide was produced by the oxidation, and the oxidation products of dopamine were highly toxic to the marine diatom Nitzschia closterium. Hydrogen peroxide and the superoxide radical did not oxidize dopamine at physiological pH. Some electrophilic compounds, including ascorbic acid, dehydroascorbic acid and thiamine, effectively inhibited dopamine oxidation. The Groote Eylandt Aborigines are likely to be deficient in ascorbic acid (vitamin C) and thiamine (vitamin B 1), and these deficiencies, as well as their lifestyle, may predispose them to manganese intoxication.

A novel adsorbent for the determination of the toxic fraction of copper in natural waters

A novel adsorbent for the determination of the toxic fraction of copper in natural waters is described. Aluminium hydroxide adsorbed on a sulfonic acid cation-exchange resin quantitatively retains copper(II) ions in the absence of organic ligands such as fulvic, humic and tannic acids. In the presence of these ligands, a smaller fraction of copper is adsorbed and can be related to the toxic fraction. The toxic fraction determined by this method agreed well with results of algal assay with the marine diatom Nitzschia closterium in seawater and the green alga Chlorella pyrenoidosa in a synthetic soft water. The aluminium hydroxide-coated column also quantitatively adsorbs lipid-soluble copper complexes, which can be leached selectively from the column with methanol. The Al:OH ratio on the resin was 1:2 and the conditional stability constants (log K) of the Cu 2+-adsorbent complexes in seawater and synthetic soft water were found to be 9.87 and 11.10, respectively; these values are similar to the equilibrium constatns for the reaction between Cu 2+ and algae. The application of this adsorbent in an in-situ instrument for the continuous, unattended determination of the toxic fraction of copper and some other toxic metals in natural waters is outlined.

Reversibility of copper-thiol binding in Nitzschia closterium and Chlorella pyrenoidosa

The effect of ionic copper and copper complexes on intracellular thiols in the marine diatom Nitzschia closterium (Ehrenberg) W. Smith, and the green alga Chlorella pyrenoidosa Chick was studied using the lipid-soluble thiol reagent, dithiodipyridine. Treatment of cells with copper ions significantly reduced the concentration of intracellular thiol groups, whereas copper complexes and H 2O 2 had no effect. To test the reversibility of intracellular copper-thiol binding in N. closterium and C. pyrenoidosa, cells exposed to copper (60–200 μg Cu·1 −1) were treated with various sulfhydryl compounds and chelating agents, with and without removal of copper from solution. Cysteine, glutathione (GSH), EDTA and bathocuproine disulfonate were most effective in reversing copper toxicity, whereas oxidized glutathione, cystine and glycine had no effect. It is proposed that copper (II) adsorbed to the algae is reduced to copper (I) by -SH groups, and bound as a Cu(I)-S-complex. Sulfhydryl compounds can reverse toxicity through thiol-disulfide interchange reactions, allowing algal growth to recommence, and powerful copper-chelating agents produce the same effect by withdrawing copper from the cell.

Toxicity of copper complexes to the marine diatom Nitzschia closterium

Copper ions initially bind to Nitzschia with a stability constant, β 1, in the range log β 1 < 13 > 10, a value which precludes sulfur bonding and suggests involvement of a membrane protein. Although the algal growth rate in sea water was halved by 20 μg Cu·1 −1, photosynthesis was not affected until the copper concentration was above 100 μg Cu·1 −1. This decoupling of growth and photosynthesis did not occur to the same extent with other algae species tested, and it is proposed that it results from an intracellular reaction between Cu 2+ and GSH, leading to a lowering of the GSH/GSSG ratio and suppression of mitosis. Water-soluble ligands generally decreased the toxicity of copper, whereas lipid-soluble copper complexes were highly toxic. Oxine, 2,9-dimethyl-1,10-phenanthroline, and pyridyl- and thiazolyl-hydroxyazo compounds formed exceptionally toxic complexes. The toxicity of many redox-active compounds and their copper complexes appears to be due, at least partly, to the intracellular generation of hydrogen peroxide during oxidation of these compounds, an effect exacerbated by the ability of copper to inhibit catalase. Hydrogen peroxide is highly toxic towards marine algae, and may be a natural growth-inhibiting factor. Hydroxyl radicals (OH·), generated intracellularly from a mixture of hydrogen peroxide and the lipid-soluble thiol, cysteine, were also extremely toxic. However, when OH· or superoxide radicals O 2 sce; were generated extracellularly, no effect on growht rate was observed, even though singlet oxygen ( 1O 2) did seriously reduce growth.

A new algal assay procedure for the determination of the toxicity of copper species in seawater

Abstract Algal assays using the marine diatom Nitzschia closterium were carried out in raw, unenriched seawater to avoid the complexlng of copper by silicate, colloidal ferric hydroxide and other compounds which occurs in standard culture media. The algae remained in logarithmic growth for at least 72 h in unenriched seawater. The advantages of using this simple medium for the study of the effect of copper speciation on toxicity are outlined. Copper ions inhibit the growth of Nitzschia, but not the photosynthesis; possible reasons for this decoupling of cell division and the photosynthesis apparatus are discussed.

Evaluation of some physico-chemical techniques for the determination of the fraction of dissolved copper toxic to the marine diatom nitzschia closterium

The toxicity of copper to the marine diatom Nitzschia closterium was determined by growth rate measurements in the presence of a range of copper complexing agents, both natural and synthetic. The measurements were made in raw, unenriched sea water to avoid the reaction of copper with silicate or colloidal hydrated iron(III) oxide which occurs in standard culture media. The algae remained in exponential growth for at least 72 h in unenriched sea water and, in the presence of copper, produced an exudate which decreased the concentration of labile copper. Labile copper was measured in the algal assay solutions by using anodic stripping voltammetry (a.s.v.) at different deposition potentials, by separation on iminodiacetate (Chelex-100) and thiol resins, and by extraction with hexane/n-butanol (9:1) to simulate lipid solubility. No consistent correlation was observed between the toxic fraction of copper measured by algal assay, and the labile copper determined by the physico-chemical techniques. Although some of the naturally-occurring ligands (e.g., fulvic acid and iron—humic acid colloid) gave reasonable agreement between the toxic fraction and a.s.v.-labile copper, the chelating resins usually grossly overestimated toxicity. Lipid-soluble complexes of copper with synthetic ligands (e.g., 8-quinolinol and diethyldithiocarbamate) were highly toxic; as little as 2 μg Cu l −1 in the presence of 5 × 10 −5 M 8-quinolinol caused complete depression of algal growth. It is proposed that the extreme toxicity of lipid-soluble copper complexes results from their ability to catalyse the intercellular formation of highly destructive hydroxyl free radicals (OH.) from molecular oxygen, in a Fenton-type reaction.

The Effect of Copper, Zinc, Cobalt and Manganese on the Growth of the Marine Diatom Nitzschia closterium

RoSKO, JOHN J., and JOSEPH W. RACHLIN (Dept. Biol. Sciences, Lehman Coll., City Univ. of New York, Bronx, New York 10468). The effect of copper, zinc, cobalt and manganese on the growth of the marine diatom Nitzschia closterium. Bull. Torrey Bot. Club 102: 100-106. 1975.-ATn extension of a previously pnblished algal cell technique (Rachlin and Farran 1974) was used in this study to monitor the effects of copper, zinc, cobalt ancd manganese on the growth of the diatom Nitzschia closterium which had been isolated from the western end of Long Island Sound. The concentration of each metal which depressed the growth rate of this speeies by 50% (EC,0) after a 96 hr exposure was calculated to be 0.0329+0.00005 ppm for Cu, 0.271+0.0003 ppm for Zn, 10.2+0.0164 ppm for Co and 25.7+0.0237 ppm for Mn. The effects of chelation on the toxicity of each metal were also determined. The results of this testing procedure indicate the potential effects of heavy metal stress on primary productivity. This may be of particular concern in waters nearlarge urban centers like New York City, where heavy industrialization may discharge metal toxicants into the aquatic environment.

The origin of the marine polyunsaturated fatty acids. Composition of some marine plankton

Summary and ConclusionsShrimp, crabs, the marine diatomNitzschia closterium, Platymonas sp., and the fresh water algaChlorella pyrenoidosa were maintained or cultured in the laboratory. The crustacea were fed low‐fat, cottonseed oil, and menhaden oil rations. The fatty acid composition of all groups, as well as that of native phytoplankton and zooplankton catches, were determined as the extinction coeffcients, (E1%1cm.), at wavelengths of maximum absorption.It was found that both shrimp and crabs lost much of their polyunsaturated acids on the fat‐free diet and regained it again by ingestion, as do fish. The shrimp however appeared to synthesize more highly unsaturated acids from cottonseed oil than did other aquatic animals.Phytoplankton do produce a high level of polyunsaturated fatty acids.The importance of the determination of the structure of aquatic plant and animal fatty acids in the problem of the origin of the acids and their mechan ism of synthesis was discussed.

Note on the Absorption of Organic Phosphorus Compounds by NitzschiaClosterium in the Dark

It has been observed by Chu (1946) that the marine diatom Nitzschia closterium, in bacteria-free culture, grows in the light with inositol hexaphosphate as phosphorus source, and with glycerophosphate.

On the use of antibiotics for isolating bacteria-free cultures of marine phytoplankton organisms

In the course of some studies on the kinetics of growth and the biochemical activities of a marine diatom it became desirable to obtain bacteria-free cultures. The classical method of obtaining such pure algal cultures involves either repeatedly washing single cells in sterile medium or obtaining discrete bacteria-free algal colonies by growth on a solid medium. Both these methods have been widely applied to fresh-water species by Pringsheim (1946) and others, whilst Chu (1946) has used both methods with the marine diatom Nitzschia closterium (Ehrenberg) Wm. Smith forma minutissima.