Effects of heavy metal ions (Cu2+, Pb2+ and Cd2+) on DNA damage of the gills, hemocytes and hepatopancreas of marine crab, Charybdis japonica

There are rising concerns about the hazardous effects of heavy metals on the environment. In this study, comet assay and DNA alkaline unwinding assay were conducted on the tissues (gills, hepatopancreas, and hemocytes) of Charybdis japonica in order to illustrate genotoxicity of three heavy metal ions (Cu2+, Pb2+, and Cd2+) on the marine crabs C. japonica. The crabs were exposed to Cu2+ (10, 50, and 100 ?g.L?1), Pb2+ (50, 250, and 500 ?g L?1) and Cd2+ (5, 25, and 50 ?g L?1), and the tissues were sampled at days 0.5, 1, 3, 6, 9, and 15. DNA alkaline unwinding assay was used for testing the DNA single strand break in gills and hepatopancreas and comet assay was employed for testing the DNA damage in hemocytes. The results showed that the DNA damage (F-value) of gills in the crabs exposed to the three heavy metals was decreased gradually during the exposure periods and there was a dose-time response relationship in certain time, suggesting that the levels of DNA single strand break in all the experimental groups increased significantly compared to the controls. Changes of F-value in hepatopancreas of the crabs exposed to the three heavy metals were similar to those in gills except that the peak values were found in the 500 ?g L?1 Pb2+ treatment group at day 3 and the 50 ?g L?1 Cd2+ treatment group at day 9. The ranks of DNA damage in gills and hepatopancreas induced by the three heavy metal ions (50 ?g L?1, day 15) were Cd2+ >Pb2+ >Cu2+ and Pb2+ >Cu2+ >Cd2+. The levels of DNA damage in gills were higher than those in hepatopancreas in the same experimental group. It can be concluded that indices of DNA damage can be used as the potential biomarkers of heavy metal pollution in marine environment.     

Effects of heavy metals （Pb^2＋ and Cd^2＋） on the ultrastructure, growth and pigment contents of the unicellular cyanobacterium Synechocystis sp. PCC 6803

The unicellular cyanobacterium Synechocystis sp. PCC 6803, a model organism known for its unique combination of highly desirable molecular genetic, physiological and morphological characteristics, was employed in the present study. The species was cultured in BG11 liquid medium contained various initial concentrations of Pb²⁺ and Cd²⁺ (0, 0.5, 1, 2, 4, 6 and 8 mg/L). The experiment was conducted for six days and the metal induced alterations in the ultrastructure, growth and pigment contents were assessed. Alterations in the ultrastructure of the Synechocystis sp. PCC 6803 cells became evident with the increased (>4 mg/L Pb²⁺) metal concentration. The photosynthetic apparatus (thylakoid membranes) were found to be the worst affected. Deteriorated or completely destroyed thylakoid membranes have made large empty spaces in the cell interior. In addition, at the highest concentration (8 mg/L Pb²⁺), the polyphosphate granules became more prominent both in size and number. Despite the initial slight stimulations (0.2, 3.8 and 6.5% respectively at 0.5, 1 and 2 mg/L Pb²⁺), both metals inhibited the growth in a dose-dependent manner as incubation progressed. Pigment contents (chlorophyll α, β carotene and phycocyanin) were also decreased with increasing metal concentration. Cells exposed to 6 mg/L Pb²⁺, resulted in 36.56, 37.39 and 29.34% reductions of chlorophyll α, β carotene and phycocyanin respectively over the control. Corresponding reductions for the same Cd²⁺concentrations were 57.83, 48.94 and 56.90%. Lethal concentration (96 h LC₅₀) values (3.47 mg/L Cd²⁺ and 12.11 mg/L Pb²⁺) indicated that Synechocystis sp. PCC 6803 is more vulnerable to Cd²⁺ than Pb²⁺. Supported by the Chinese Scholarship Council     

TOXIC EFFECTS OF Zn~(2+), Cu~(2+), Cd~(2+)and NH_3 ON CfflNESE PRAWN

This study deals with the toxic effects of Zn2+, Cu2+, Cd2+ and NH3 on Chinese prawn (Penaeus orientalis). The median lethal concentrations of the toxic matters on Chinese prawn were determined. Cd2+>Cu2+>Zn2+>NH3-Nm is the toxic order of these chemicals to Chinese prawn. The toxic order of concentrated Zn2+ and Cd2+ in tissues and organs of Chinese prawn is in the order gill > shell + appendage>viscera > muscle..     

Variations of antioxidant enzyme activity and malondialdehyde content in nemertean Cephalothrix hongkongiensis after exposure to heavy metals

The antioxidant enzyme activity and malondialdehyde (MDA) content of Cephalothrix hongkongiensis were studied to assess variations in the biochemical/physiological parameters of nemerteans under heavy metal stress. Worms were exposed to copper, zinc and cadmium solutions at different concentrations, and the activity of three antioxidant enzymes, catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPX), and MDA content were measured. The results show that the activity of each enzyme changed immediately after exposure to heavy metals. CAT was invariably inhibited throughout the experimental period, while the SOD activity was significantly elevated by exposure to Cu²⁺ for 48 h, but then decreased. SOD was inhibited by Zn²⁺during the first 12 h of exposure, but activated when exposed for longer periods. Under Cd²⁺ stress, SOD activity decreased within 72 h. GPX activity varied greatly, being significantly increased by both Cu²⁺ and Zn²⁺, but significantly inhibited by Cd²⁺ in the first 12–24 h after exposure. MDA content increased on Cu²⁺ exposure, but normally decreased on Zn²⁺ exposure. MDA content followed an increase-decrease-increase pattern under Cd²⁺ stress. In conclusion, the antioxidant system of this nemertean is sensitive to heavy metals, and its CAT activity may be a potential biomarker for monitoring heavy metal levels in the environment.     

Toxic effects of zinc on four species of freshwater fish

The toxic effects of Zn²⁺ on Silver Carp (Hypophthalmichthyps molitrix C. et V.), Big Head Carp (Aristrichthys nobilis Richardson), Grass Carp (Ctenopharyngodon ilellus C. et V.), and Blunt Snout Bream (Megalobrama aimbly-cephala Yih) are studied. The test results are: (1) There are linear correlations between 24h LC₅₀ and 48h LC₅₀ of Zn²⁺ for Silver Carp fingerling and temperature. 24h LC₅₀, 48h LC₅₀ and 96h LC₅₀ of Zn²⁺ for fry of the four species are also determined; (2) There are logarithmic correlations between the growth rates of the fry and the concentrations of Zn²⁺ and between expansion of fish egg membranes after absorbing water and concentrations of Zn²⁺; (3) The tolerance of fry of the four species to Zn²⁺ is in the following order: Grass Carp>Silver Carp>Blunt Snout Bream>Big Head Carp; (4) The safe concentrations of Zn²⁺ are: Big Head Carp: 0.008 mg/L, Grass Carp: 0.046 mg/L, Blunt Snout Bream: 0.010 mg/L, Silver Carp: 0.012 mg/L, Silver Carp fingerling: 0.09 mg/L.     

Bioaccumulation of Pb<Superscript>2+</Superscript> and its effects on growth,morphology and pigment contents of <Emphasis Type="Italic">Spirulina</Emphasis> (<Emphasis Type="Italic">Arthrospira</Emphasis>) <Emphasis Type="Italic">platensis</Emphasis>

A laboratory experiment was conducted to assess the bioaccumulation of Pb²⁺ and its effects on growth, morphology and pigment contents of Spirulina (Arthrospira) platensis. The specimen cultured in Zarrouk liquid medium was treated with various initial metal concentrations (0, 5, 10, 30, 50 and 100 μg mL⁻¹). The growth of S. platensis was adversely affected by Pb²⁺ at high concentrations (30, 50 and 100 μg mL⁻¹). However, at low concentrations (5 μg mL⁻¹), Pb²⁺ could stimulate its growth slightly. The pigment contents (chlorophyll α and β carotene) were decreased in a dose-dependent manner. The highest reductions (67% and 53% respectively in chlorophyll α and β carotene) were observed in 100 μg mL⁻¹ treatment group. The LC₅₀ (96 h) of Pb²⁺ was measured as 75.34 μg mL⁻¹. Apart from a few cases of filament breakages at elevated concentrations (50 and 100 μg mL⁻¹), morphological abnormalities are not specific. Metal bioaccumulation increased with Pb²⁺ concentrations, but decreased with exposure time. The maximum accumulated amount was 188 mg g⁻¹ dry weight. The bioconcentration factor (BCF) reached to a peak at day 2, followed by a gradual reduction for all the exposure concentrations. S. platensis is able to tolerate considerably high Pb²⁺ concentrations. Consequently it can be used as a potential species to remove heavy metal from contaminated waters.     

Effects of Na+/K+ ratio of groundwaters on the gill ion-transport enzyme activity, plasma osmolality and growth of Cynoglossus semilaevis juveniles

The effects of environmental Na⁺/K⁺ ratio on the gill ion-transport enzyme activity, plasma osmolality and growth of Cynoglossus semilaevis juveniles were investigated. The results showed that, plasma osmolality was similar among flounder adapted to different Na⁺/K⁺ ratios of saline groundwaters (P>0.05), while the growth, gill Na⁺, K⁺-ATPase and HCO₃ ⁻-ATPase activities were affected by Na⁺/K⁺ ratio significantly (P<0.05). The gill Na⁺, K⁺-ATPase activity reached its maximum on day 3, then decreased gradually from day 3 to day 9 and remained constant from day 9 to day 15. The peak values of gill Na⁺, K⁺-ATPase activity were detected on day 3 for all Na⁺/K⁺ ratios of saline groundwaters, then the enzyme activities descended, and on day 9 the enzyme activities achieved steady state, and the gill HCO₃ ⁻-ATPase activity increased rapidly and achieved steady state after one day. During steady state, the gill Na⁺, K⁺-ATPase and HCO₃ ⁻-ATPase activity of Na⁺/K⁺ ratios 20 and 40 treatments were significantly higher than those in the control group (Na⁺/K⁺ ratio 27.5), while there were no significant differences between the Na⁺/K⁺ ratio 30 treatment and the control group; the gill Na⁺, K⁺-ATPase activity of Na⁺/K⁺ ratio 20 and 40 treatments were significantly higher than that for ratio 30 treatment, but there were no significant differences of gill HCO₃ ⁻-ATPase activity among these treatments. At the end of the 15-day experiment, the weight gain (%) and specific growth rate (SGR) of flounders maintained in seawater were significantly higher than those in groundwaters; significant differences also occurred among the treatments; Na⁺/K⁺ ratio 30 treatment had the highest values (33.7% and 1.94 respectively), which were significantly higher than those under Na⁺/K⁺ ratios 20 and 40 treatments. Therefore, for the saline groundwater used in this experiment, it is suggested that the Na⁺/K⁺ ratio be adjusted to approximately 30, i.e., as close to that of natural seawater as possible in the culture of flounder.     

Impacts of CO<Subscript>2</Subscript> enrichment on growth and photosynthesis in freshwater and marine diatoms

The physiological responses of Nitzschia palea Kvtzing, a freshwater diatom, to elevated CO2 were investigated and compared with those of a marine diatom, Chaetoceros muelleri Lemmermann previously reported. Elevated CO2 concentration to 700 μl/L increased the dissolved inorganic carbon （D!C） and lowered the pH in the cultures of N. palea, thus enhancing the growth by 4%-20% during the whole growth period. High CO2-grown N. palea cells showed lower levels of dark respiration rates and higher Ik values. Light-saturated photosynthetic rates and photosynthetic efficiencies decreased in N. palea with the doubling CO2 concentration in airflow to the bottom of cultures, although the doubling CO2 concentration in airflow to the surface cultures had few effects on these two photosynthetic parameters. N. palea cells were found to be capable of using HCO5 in addition to gaseous CO2, and the CO2 enrichment decreased their affinity for HCO5 and CO2. Although doubled CO2 level would enhance the biomass of N. patea and C. muelleri to different extents, compared with the marine diatom, it had a significant effect on the specific growth rates of N. palea. In addition, the responses of photosynthetic parameters of IV. palea to doubled CO2 concentration were almost opposite to those of C. muelleri.     

Effects of composition and structure of alginates on adsorption of divalent metals

Results of a series of experiments (on the adsorption of divalent metal ions by dried alginic acid, Na and Ca alginates of different composition and block structure) conducted in this systematic study of the effects of the composition and structure of alginates on the static adsorption equilibrium of divalent metal ions indicate that the properties of alginate adsorption to divalent metal ions are highly different, depending not only on the cations used, but also on the form and structure of the alginates. There is close correlation between the adsorption properties and the structure of the alginates. The selectivity coefficient of Na alginate for Cd-Sr ion exchange tends to increase with the increase of theM/G ratio in alginate, whereas the adsorption capacity of Ca alginate for Cu²⁺ ion decrease with the increase of the G-block or the average length of the G-block and the total adsorption capacity of alginic acid is found to vary in the same order as theF _MM(diad frequency) in alginate in the mixed solution of Sr²⁺, Ba²⁺ and Cd²⁺. Contribution No. 2166 from the Institute of Oceanology, Chinese Academy of Sciences.     

The influence of Fe^2＋ on growth and development of cells enzymatically isolated from Porphyra yezoensis blades

Fe^2＋ acted as an accessorial factor for many cellular enzymatic reactions is very important for seaweed growth and development, but the Fe^2＋ requirement in nori had not been seen. Porphyra yezoensis cells were separated enzymatically and cultured in a series of sterilized seawater media containing various concentrations of Fe^2＋. The growth development and cell were investigated in this work. Through this experiment, two biologically-meant concentration scales were found, one is low concentrations, 12.1-102.1μg/L, 10-100 times than that in seawater, favoring the development of isolated cells of Porphyra and the other was high concentrations, more than 10mg/L inhibiting the cell growth, leading to the deformity and shrinkage of the cells. At the concentration of 50 mg/L, the cells stopped growing and died eventually.     

Study on the relationship between speciation of heavy metals and their ecotoxicity

Heavy metal is a main pollutant in the marine ecosystem, so study on the effect of heavy metal on phytoplankton is important. Algae (Chaetoceros sp.,Dunaliella sp.,Dicrateria zhanjiangenis Hu. var. sp.) were laboratory cultured to observe the effect of heavy metals on their growth. The effect of different metal ion concentration, the detoxication effect of complexation agents and the growth of algae in different media and different nutrition levels were studied to evaluate the effect of metal speciation. It is proved that trace amount of heavy metals can stimulate the growth of algae cells but that high concentration is lethal. The sequence of toxicity is Cd²⁺>Zn²⁺>Pb²⁺. In ordinary nutrition conditions, the detoxication sequence of complexation agents toChaetoceros sp. is EDTA >sodium salicylate>sodium oxalate >sodium citrate>sulfanilic acid>O-phenanthroline. This is in good conformity with the stability constant sequence of these agents with copper and good evidence that toxicity of metal ion is related to its activity and not to its total concentration. Project supported by the Fund of Excellent Young Teachers of State Educational Commission of China.     

Estimate of global sea-air CO<Subscript>2</Subscript> flux with sea-state-dependent parameterization

Although the annual global sea-air CO₂ flux has been estimated extensively with various wind-dependent-k parameterizations, uncertainty still exists in the estimates. The sea-state-dependent-k parameterization is expected to improve the uncertainty existing in these estimates. In the present study, the annual global sea-air CO₂ flux is estimated with the sea-state-dependent-k parameterization proposed by Woolf (2005), using NOAA/NCEP reanalysis wind speed and hindcast wave data from 1998 to 2006, and a new estimate, −2.18 Gt C year⁻¹, is obtained, which is comparable with previous estimates with biochemical methods. It is interesting to note that the averaged value of previous estimates with various wind-dependent-k parameterizations is almost identical to that of previous estimates with biochemical methods by various authors, and that the new estimate is quite consistent with these averaged estimates.     

FOOD SOURCES AND CARBON BUDGET OF CHINESE PRAWN PENAEUS CHINENSIS

This study deals with contribution of artificial food pellet and natural food to Chinese prawn (Penaeus orientalis) growth in a semiintensive culture pond. The prawn carbon consumption, budget, and the effects of some factors on the budget were investigated. The results showed that 26.2% of P. orientalis growth carbon came from formulated feed at the initial culture stage (when the prawns were 0.06±0.01 g in wet weight), and was 62.5% when the prawns were 9.56±1.04 g. The remaining part of the growth carbon was derived from organic fertilizer and natural food. The highest growth rate occurred at 20×10-3 salinity. Suitable salinity for culturing Chinese prawn was (20-28)×10-3.     

The effects of nitrogen,phosphorus, vitamins and trace metals on the growth of the red tide organismProrocentrum Micans

Laboratory culture experiments showed that <100μ mol/L nitrate, amonium or mixture of amino acids promote the growth of the red tide organismProrocentrum micans Ehrenb, but that >100μmol/L of ammonium, or mixture of glycine and glutamate was harmful to growth, and that orthophosphate wasP. micans’ main phosphorous source in the ocean. Presence of 80μ mol/L EDTA, 0.5 to 1 μmol/L Fe³⁺, 1.0 to 20.0 μ mol/L Mn²⁺ 0.1 to 0.4 μmol/L Co²⁺ in the culture medium could improve the growth ofP. micans. Vitamin B₁ promoted growth, but vitamin B₁₂ and biotin did not. The estimated minimum cell quotas (q _o) for nitrogen and phosphorus being 0.74 pmole/cell and 0.045 pmole/cell show that phosphorus (more than nitrogen) limits the growth ofP. micans in the study area. This project was supported by the Natural Science Foundation of Zhejiang Province.     

A multilayer study of pCO2 in the surface waters of the Yellow and South China Seas in spring and the sea-air carbon dioxide flux

A multilayer study of pCO₂ for the Yellow and South China Seas in the surface waters was conducted based on data from four cruises sponsored by the China SOLAS Project in 2005 and 2006, including data for the surface microlayer (SML), subsurface layer (SSL) and surface layer (SL). The carbon fluxes across the air-sea interface were calculated. The results showed that the pCO₂ values in the surface waters of the study area decreased in the following order: pCO_{2 SML} > pCO_{2 SSL} > pCO_{2 SL}. The highest values were found in March for all SML, SSL and SL, followed by those in April, and the lowest were in May. The pCO₂ values had a significant positive correlation with temperature or salinity. While there was no relationship between pCO₂ and longitude, there was a significant negative correlation between it and latitude, i.e., ‘high latitude low pCO₂’. By using four calculation models, the carbon dioxide fluxes ( ) in spring in the Yellow and South China Seas, which were found to act as a ‘sink’ of atmospheric CO₂, were preliminarily estimated on the basis of the pCO₂ data in the SML to be −7.00×10⁶t C and −22.35×10⁶t C, respectively. It is suggested that the calculated on the basis of pCO₂ data in the SML is more reliable than that calculated on the basis of those in the SL.     

A sulfotransferase specific to N-21 of gonyautoxin 2/3 from crude enzyme extraction of toxic dinoflagellate Alexandrium tamarense CI01

Sulfotransferase (ST) is the first enzyme discovered in association with paralytic shellfish poisoning (PSP) toxin biosynthesis in toxic dinoflagellates. This study investigates the ST activity in crude enzyme extraction of a toxic dinoflagellate species, Alexandrium tamarense CI01. The results show that crude enzyme can transfer a sulfate group from 3’-phosphoadenosine 5’-phosphosulfate (PAPS) to N-21 in the carbamoyl group of gonyautoxin 2/3 (GTX2/3) to produce C1/C2, but is inactive toward STX to produce GTX5. The crude enzyme is optimally active at pH 6.0 and 15°C. The activity is enhanced by Co2 , Mg2 , Mn2 and Ca2 individually, but is inhibited by Cu2 . Moreover, the activity shows no difference when various sulfur compounds are used as sulfate donors. These results demonstrate that the ST specific to GTX2/3 is present in the cells of A. tamarense CI01 and is involved in PSP toxin biosynthesis. In addition, the ST from different dinoflagellates is species-specific, which explains well the various biosynthesis pathways of the PSP toxins in toxic dinoflagellates.     

Effects of iron electrovalence and species on growth and astaxanthin production of Haematococcus pluvialis

To increase the cell concentration and the accumulation of astaxanthin in the cultivation of Haematococcus pluvialis, effects of different iron electrovalencies (Fe²⁺-EDTA and Fe³⁺-EDTA) and species (Fe-EDTA, Fe(OH)_x^32x and FeC₆H₅O₇) addition on cell growth and accumulation of astaxanthin were studied. Results show that different iron electrovalencies have various effects on cell growth and astaxanthin accumulation of H. pluvialis. Compared with Fe³⁺-EDTA, Fe²⁺-EDTA stimulate more effectively the formation of astaxanthin. The maximum astaxanthin content (30.70 mg/g biomass cell) was obtained under conditions of 18 μmol/L Fe²⁺-EDTA, despite the lower cell density (2.3×10⁵ cell/ml) in such condition. Fe³⁺-EDTA is more effective than Fe²⁺-EDTA in improving the cell growth. Especially, the maximal steady-state cell density, 2.9×10⁵ cell/ml was obtained at 18 μmol/L Fe³⁺-EDTA addition. On the other hand, all the various species of iron (EDTA-Fe, Fe(OH)_x^32x, FeC₆H₅O₇) are capable to improve the growth of the algae and astaxanthin production. Among the three iron species, FeC₆H₅O₇ performed the best. Under the condition of a higher concentration (36 μmol/L) of FeC₆H₅O₇, the cell density and astaxanthin production is 2 and 7 times higher than those of iron-limited group, respectively. The present study demonstrates that the effects of the stimulation with different iron species increased in the order of FeC₆H₅O₇, Fe(OH)_x^32x and EDTA-Fe. Supported by Xiamen Scientific and Technologic Projects (No. 3052Z20031086), Xiamen University Alumni Association Foundation in Singapore, and the First National College Students Innovative Experimental Project     

H2 PRODUCTION BY ANABAENA VARIABILIS MUTANT IN COMPUTER CONTROLLED TWO-STAGE AIR-LIFT TUBULAR PHOTOBIOREACTOR

A 4.34 liter two-stage air-lift photobioreactor incorporatingAnabaena variabilis ATCC29413 mutant PK84 was used to study H₂ production. Results showed that H₂ production increased with increasing light intensity from 47 μE/(m²·s) up to 190 μE/(m²·s), but that further increase of light intensity decreased the H₂ production because of the inhibition due to the high pO₂. The data also indicated that longer argon gas charge resulted in more H₂ produced due to the increase of nitrogenase activities and heterocyst frequency, and that more than 1.3 L net H₂ was produced from this computer controlled photobioreactor.     

CHEMICAL CHARACTERISTICS OF FOUR KINDS OF WATER IN THE RONGBUK GLACIER BASIN, MOUNT QOMOLANGMA

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Effects of Mg2+, NaCl, citric acid, and other factors on synthesis and accumulation of β-carotene inDunaliella salina

The effect of Mg²⁺, NaCl and citric acid on the accumulation of β-carotene inDunaliella Salina was studied. The experimental results showed that 10.5 mmol/L Mg²⁺, 5 mol/L NaCl, 3 μmol/L citric acid, and CO₂ are favorable forDunaliella Salina cell growth and β-carotene accumulation. After 144 h culture under the above conditions, theDunaliella Salina biomass increased by 7.18 times; β-carotene reached 9.61%. Project 89023990 supported by the Shandong Natural Science Fund.