Comparison of Toxicity and Cellular Uptake of CdSe/ZnS and Carbon Quantum Dots for Molecular Tracking Using Saccharomyces cerevisiae as a Fungal Model.

Plant resource sharing mediated by mycorrhizal fungi has been a subject of recent debate, largely owing to the limitations of previously used isotopic tracking methods. Although CdSe/ZnS quantum dots (QDs) have been successfully used for in situ tracking of essential nutrients in plant-fungal systems, the Cd-containing QDs, due to the intrinsic toxic nature of Cd, are not a viable system for larger-scale in situ studies. We synthesized amino acid-based carbon quantum dots (CQDs; average hydrodynamic size 6 ± 3 nm, zeta potential -19 ± 12 mV) and compared their toxicity and uptake with commercial CdSe/ZnS QDs that we conjugated with the amino acid cysteine (Cys) (average hydrodynamic size 308 ± 150 nm, zeta potential -65 ± 4 mV) using yeast Saccharomyces cerevisiae as a proxy for mycorrhizal fungi. We showed that the CQDs readily entered yeast cells and were non-toxic up to 100 mg/L. While the Cys-conjugated CdSe/ZnS QDs were also not toxic to yeast cells up to 100 mg/L, they were not taken up into the cells but remained on the cell surfaces. These findings suggest that CQDs may be a suitable tool for molecular tracking in fungi (incl. mychorrhizal fungi) due to their ability to enter fungal cells.

LuxCDABE--transformed constitutively bioluminescent Escherichia coli for toxicity screening: comparison with naturally luminous Vibrio fischeri.

We show that in vitro toxicity assay based on inhibition of the bioluminescence of recombinant Escherichia coli encoding thermostable luciferase from Photorhabdus luminescens is a versatile alternative to Vibrio fischeri Microtox™ test. Performance of two luxCDABE-transformed E. coli MC1061 constructs (pDNlux) and (pSLlux) otherwise identical, but having 100-fold different background luminescence was compared with the performance of V. fischeri. The microplate luminometer and a kinetic Flash-Assay test format was used that differently from Microtox test is also applicable for high throughput analysis. Toxic effects (30-s till 30-min EC(50)) of four heavy metals (Zn, Cd, Hg, Cu) and three organic chemicals (aniline, 3,5-dichloroaniline and 3,5-dichlorophenol) were studied. Both E. coli strains had comparable sensitivity and the respective 30-min EC(50) values highly correlated (log-log R(2) = 0.99; p < 0.01) showing that the sensitivity of the recombinant bacteria towards chemicals analyzed did not depend on the bioluminescence level of the recombinant cells. The most toxic chemical for all used bacterial strains (E. coli, V. fischeri) was mercury whereas the lowest EC(50) values for Hg (0.04-0.05 mg/L) and highest EC(50) values for aniline (1,300-1,700 mg/L) were observed for E. coli strains. Despite of that, toxicity results obtained with both E. coli strains (pSLlux and pDNlux) significantly correlated with V. fischeri results (log-log R(2) = 0.70/0.75; p < 0.05/0.01). The use of amino acids (0.25%) and glucose (0.05%)-supplemented M9 medium instead of leucine-supplemented saline significantly (p < 0.05) reduced the apparent toxicity of heavy metals to both E. coli strains up to three orders of magnitude, but had little or no complexing effect on organic compounds. Thus, P. luminescens luxCDABE-transformed E. coli strains can be successfully used for the acute toxicity screening of various types of organic chemicals and heavy metals and can replace V. fischeri in certain cases where the thermostability of luciferase >30 °C is crucial. The kinetic Flash Assay test format of the bioluminescence inhibition assay facilitates high throughput analysis. The assay medium, especially in case of testing heavy metals should be a compromise: optimal for the viability/luminescence of the recombinant test strain and of minimum complexing potential.

Antibacterial Activity of Positively and Negatively Charged Hematite (α-Fe2O3) Nanoparticles to Escherichia coli, Staphylococcus aureus and Vibrio fischeri.

In the current study, the antibacterial activity of positively and negatively charged spherical hematite (α-Fe2O3) nanoparticles (NPs) with primary size of 45 and 70 nm was evaluated against clinically relevant bacteria Escherichia coli (gram-negative) and Staphylococcus aureus (gram-positive) as well as against naturally bioluminescent bacteria Vibrio fischeri (an ecotoxicological model organism). α-Fe2O3 NPs were synthesized using a simple green hydrothermal method and the surface charge was altered via citrate coating. To minimize the interference of testing environment with NP's physic-chemical properties, E. coli and S. aureus were exposed to NPs in deionized water for 30 min and 24 h, covering concentrations from 1 to 1000 mg/L. The growth inhibition was evaluated following the postexposure colony-forming ability of bacteria on toxicant-free agar plates. The positively charged α-Fe2O3 at concentrations from 100 mg/L upwards showed inhibitory activity towards E. coli already after 30 min of contact. Extending the exposure to 24 h caused total inhibition of growth at 100 mg/L. Bactericidal activity of positively charged hematite NPs against S. aureus was not observed up to 1000 mg/L. Differently from positively charged hematite NPs, negatively charged citrate-coated α-Fe2O3 NPs did not exhibit any antibacterial activity against E. coli and S. aureus even at 1000 mg/L. Confocal laser scanning microscopy and flow cytometer analysis showed that bacteria were more tightly associated with positively charged α-Fe2O3 NPs than with negatively charged citrate-coated α-Fe2O3 NPs. Moreover, the observed associations were more evident in the case of E. coli than S. aureus, being coherent with the toxicity results. Vibrio fischeri bioluminescence inhibition assays (exposure medium 2% NaCl) and colony forming ability on agar plates showed no (eco)toxicity of α-Fe2O3 (EC50 and MBC > 1000 mg/L).

Impact of surface functionalization on the toxicity and antimicrobial effects of selenium nanoparticles considering different routes of entry.

Selenium nanoparticles (SeNPs) were first designed as nutritional supplements, but they are attractive also for use in diagnostic and therapeutic systems owing to their biocompatibility and protective effects. This study aimed to examine if different SeNPs stabilization strategies affect their (i) antimicrobial activity against bacteria Escherichia coli and Staphylococcus aureus and yeast Saccharomyces cerevisiae and (ii) toxicity to human cells of different biological barriers i.e., skin, oral and intestinal mucosa. For surface stabilization, polyvinylpyrrolidone (PVP), poly-L-lysine (PLL) and polyacrylic acid (PAA) were used rendering neutral, positively and negatively charged SeNPs, respectively. The SeNPs (primary size ~80 nm) showed toxic effects in human cells in vitro and in bacteria S. aureus, but not in E. coli and yeast S. cerevisiae. Toxicity of SeNPs (24 h IC50) ranged from 1.4 to >100 mg Se/L, depending on surface functionalization (PLL > PAA > PVP) and was not caused by ionic Se. At subtoxic concentrations, all SeNPs were taken up by all human cell types, induced oxidative stress response and demonstrated genotoxicity. As the safety profile of SeNPs was dependent not only on target cells (mammalian cells, bacteria, yeast), but also on surface functionalization, these aspects should be considered during development of novel SeNPs-based biomedical products.

Stability and toxicity of differently coated selenium nanoparticles under model environmental exposure settings.

This study, motivated to fill the knowledge gap on environmental safety of selenium nanoparticles (SeNPs), provides information on the stability and environmental safety of four differently coated SeNPs rendering both positive and negative surface charges. The stability and dissolution behaviour of SeNPs were determined in an aquatic model media of different ionic strength to provide information regarding the environmental fate of SeNPs in different environmental conditions. The environmental safety of SeNPs was evaluated by acute regulatory toxicity tests using Daphina magna and Vibrio fischeri as model organisms. Agglomeration was observed for all studied SeNPs in test media with higher ionic strength caused by the disruption of surface charge leading to electrostatic instability. Toxicity of SeNPs on both aquatic species was dose-dependent and increased with exposure time. The obtained data indicated that all of the tested SeNPs could be classified as harmful to the natural bacteria V. fischeri and harmful to toxic to crustaceans D. magna, but dependent on the coating agent used for SeNPs stabilization. Although SeNPs have attracted great interest for use in biomedicine, this study demonstrated that their ecotoxicological effects should be considered during the design of new of SeNPs-based products.

Hazard evaluation of polystyrene nanoplastic with nine bioassays did not show particle-specific acute toxicity.

Plastic is a wide-spread pollutant and must be evaluated for potential adverse effects of its breakdown product, microplastic (≤5 mm) along with its subfraction, nanoplastic (1-100 nm). Risk assessment of pollutants cannot be conducted without their toxicity (dose-response) data. In this study, toxicity of polystyrene nanoplastics (PS-NPL) was evaluated using 8 acute and 1 subchronic toxicity assays with 10 organisms of different biological complexity (bacteria, yeast, algae, protozoans, mammalian cells in vitro, crustaceans, midge larvae). Commercial 26 and 100 nm carboxylated PS-NPL spheres were chosen as model and tested in nominal concentrations up to 100 mg/L (1.025·1016 26 nm and 1.83·1014 100 nm particles/L). In most of the assays, both PS-NPL proved non-toxic (L(E)C50 > 100 mg/L) but three tests (V. fischeri, R. subcapitata, D. magna) flagged toxicity in 'as received' 26 nm PS-NPL and D. magna also in 100 nm PS-NPL (EC50 ranging from 13 to 71 mg/L). As, according to manufacturers, both PS-NPL suspensions contained additives (surfactants and biocidal NaN3), the three toxicity tests were repeated also on dialysed PS-NPL and on NaN3. Non-toxicity of dialysed PS-NPL indicated that the toxicity of 'as-received' PS-NPL was not particle-specific but false positive due to water-soluble additives in the PS-NPL preparations. NaN3 was very toxic to D. magna (48 h EC50 = 0.05 ± 0.03 mg NaN3/L), toxic to R. subcapitata (72 h EC50 = 4.97 ± 3.7 mg NaN3/L) and non-toxic to V. fischeri. Toxicity of 'as-received' PS-NPL was not fully explainable by NaN3 but also attributable to other additives in the suspensions. Toxicity research of microplastic using commercial model particles must always consider the potential influence of additives, e.g. test the toxicity of dialysed NPL for comparison. In our study, D. magna, R. subcapitata and V. fischeri were the most sensitive to PS-NPL water-soluble additives and flagged their presence in NPL preparations.

Antimicrobial potency of differently coated 10 and 50 nm silver nanoparticles against clinically relevant bacteria Escherichia coli and Staphylococcus aureus.

Silver nanoparticles (nanoAg) are effective antimicrobials and promising alternatives to traditional antibiotics. This study aimed at evaluating potency of different nanoAg against healthcare infections associated bacteria: Gram-negative Escherichia coli and Gram-positive Staphylococcus aureus. A library of differently coated nanoAg of two different sizes (10 and 50 nm) were prepared using coating agents poly-L-Lysine (PLL), cetyltrimethyl-ammonium bromide (CTAB), citrate (CIT), polyvinyl-pyrrolidone (PVP), polysorbate 80 (Tween 80), and dioctyl-sodium sulfosuccinate (AOT). Stability evaluation by means of agglomeration and dissolution behaviour was performed for all nanoAg under conditions relevant for this study. Antibacterial properties of nanoAg were addressed by determining their minimal bactericidal concentrations (MBC) in deionised (DI) water to minimise the influence of silver speciation on its bioavailability. In parallel, AgNO3 was analysed as an ionic control. Studied nanoAg were efficient antimicrobials being remarkably more potent towards E. coli than to S. aureus (4 h MBC values for different nanoAg ranged from 0.08 to 5.0 mg Ag/L and 1.0-10 mg Ag/L, respectively). The toxicity of all nanoAg to S. aureus (but not to E. coli) increased with exposure time (4 h vs 24 h). 10 nm sized nanoAg released more Ag-ions and were more toxic than 50 nm nanoAg. Coating-dependent toxicity was more prominent for 50 nm nanoAg coated with Tween 80 or CTAB rendering the least toxic nanoAg. Obtained results showed that the antimicrobial effects of nanoAg were driven by shed Ag-ions, depended on target bacteria, exposure time and were the interplay of NP size, solubility and surface coating.

Rapid screening for soil ecotoxicity with a battery of luminescent bacteria tests.

A bacterial test battery, involving i) Microtox, an aquatic test, ii) the Flash assay, a soil-suspension test (with Vibrio fischeri as the test organism), and iii) the Metal Detector assay, a semi-specific aquatic test for heavy metals (with recombinant luminescent Escherichia coli), was used in a combined toxicological and chemical hazard assessment of Estonian soils sampled from a former Soviet military airfield (13 samples) and from traffic-influenced roadsides (5 samples). The soils showed slightly elevated levels of total petroleum hydrocarbons (TPH), but not of heavy metals. In most of the samples, the levels of TPH did not exceed the Estonian permitted limit values set for residential areas. Toxicity testing was performed on both fresh and dried soils, after aqueous extraction for 1 hour and 24 hours. The toxicity results obtained with the Microtox test did not significantly differ in all of the sample treatment schemes; however, it appeared that the drying and sieving of the soils increased the bioavailability of toxicants, probably due to an enlarged reactive soil surface area. According to chemical analysis of the soils and the data from the Microtox test and the Metal Detector assay (performed on aqueous elutriates of the soils), these soils would not be considered to be hazardous. In contrast, the Flash assay performed on soil-water suspensions of dried soils, showed that most of the soils were toxic and thus probably contained undetermined particle-bound bioavailable toxicants. The photobacterial toxicity test (the Flash assay) can be recommended for the rapid screening of soils, as it is sensitive, cheap and inexpensive, and provides valuable information on particle-bound bioavailable toxicants, useful for complementing a chemical analysis and for assessing the risks originating from polluted soils.

Toxicity of Nine (Doped) Rare Earth Metal Oxides and Respective Individual Metals to Aquatic Microorganisms Vibrio fischeri and Tetrahymena thermophila.

Despite the increasing use of rare earth elements (REEs) and oxides (REOs) in various technologies, the information on their ecotoxicological hazard is scarce. Here, the effects of La3+, Ce3+, Pr3+, Nd3+, Gd3+, CeO2, and eight doped REOs to marine bacteria Vibrio fischeri and freshwater protozoa Tetrahymena thermophila were studied in parallel with REO dopant metals (Co2+, Fe3+, Mn2+, Ni2+, Sr2+). The highest concentrations of REOs tested were 100 mg/L with protozoa in deionized water and 500 mg/L with bacteria in 2% NaCl. Although (i) most REOs produced reactive oxygen species; (ii) all studied soluble REEs were toxic to bacteria (half-effective concentration, EC50 3.5-21 mg metal/L; minimal bactericidal concentration, MBC 6.3-63 mg/L) and to protozoa (EC50 28-42 mg/L); and (iii) also some dopant metals (Ni2+, Fe3+) proved toxic (EC50 ≤ 3 mg/L), no toxicity of REOs to protozoa (EC50 > 100 mg/L) and bacteria (EC50 > 500 mg/L; MBC > 500 mg/L) was observed except for La2NiO4 (MBC 25 mg/L). According to kinetics of V. fischeri bioluminescence, the toxicity of REEs was triggered by disturbing cellular membrane integrity. Fortunately, as REEs and REOs are currently produced in moderate amounts and form in the environment insoluble salts and/or oxides, they apparently present no harm to aquatic bacteria and protozoa.

Multilaboratory evaluation of 15 bioassays for (eco)toxicity screening and hazard ranking of engineered nanomaterials: FP7 project NANOVALID.

Within EU FP7 project NANOVALID, the (eco)toxicity of 7 well-characterized engineered nanomaterials (NMs) was evaluated by 15 bioassays in 4 laboratories. The highest tested nominal concentration of NMs was 100 mg/l. The panel of the bioassays yielded the following toxicity order: Ag > ZnO > CuO > TiO2 > MWCNTs > SiO2 > Au. Ag, ZnO and CuO proved very toxic in the majority of assays, assumingly due to dissolution. The latter was supported by the parallel analysis of the toxicity of respective soluble metal salts. The most sensitive tests/species were Daphnia magna (towards Ag NMs, 24-h EC50 = 0.003 mg Ag/l), algae Raphidocelis subcapitata (ZnO and CuO, 72-h EC50 = 0.14 mg Zn/l and 0.7 mg Cu/l, respectively) and murine fibroblasts BALB/3T3 (CuO, 48-h EC50 = 0.7 mg Cu/l). MWCNTs showed toxicity only towards rat alveolar macrophages (EC50 = 15.3 mg/l) assumingly due to high aspect ratio and TiO2 towards R. subcapitata (EC50 = 6.8 mg Ti/l) due to agglomeration of TiO2 and entrapment of algal cells. Finally, we constructed a decision tree to select the bioassays for hazard ranking of NMs. For NM testing, we recommend a multitrophic suite of 4 in vitro (eco)toxicity assays: 48-h D. magna immobilization (OECD202), 72-h R. subcapitata growth inhibition (OECD201), 30-min Vibrio fischeri bioluminescence inhibition (ISO2010) and 48-h murine fibroblast BALB/3T3 neutral red uptake in vitro (OECD129) representing crustaceans, algae, bacteria and mammalian cells, respectively. Notably, our results showed that these assays, standardized for toxicity evaluation of "regular" chemicals, proved efficient also for shortlisting of hazardous NMs. Additional assays are recommended for immunotoxicity evaluation of high aspect ratio NMs (such as MWCNTs).

Biotests and biosensors in ecotoxicological risk assessment of field soils polluted with zinc, lead, and cadmium.

The combined chemical and ecotoxicological hazard evaluation study was conducted on 60 smelter-influenced soils containing 1 to 13, 50 to 653, and 100 to 1,198 mg/kg of Cd, Pb, and Zn, respectively. For these soils (liquid-to-soil ratio = 10), water extractability of Zn, Cd, and Pb was less than 0.19% (median values). Acetic acid (0.11 M) extracted 23, 9.7, and 0.7% of Cd, Zn, and Pb, respectively. Although heavy metal concentrations in the studied soils were high, the toxic effects of water extracts were observed only in few samples and in few biotests (algae Selenastrum capricornutum and metal detector assay). For most of the aquatic test organisms (e.g., crustaceans, photobacteria), the bioavailable concentrations of metals in soil-water extracts were either subtoxic, or the adverse effects were compensated by soil nutrients, etc. However, analysis of the soils with recombinant Cd sensor Bacillus subtilis (pTOO24) showed that about 65% of these apparently subtoxic samples contained bioavailable Cd when analyzed in the suspension assay (detection limit 1.5 mg Cd/kg soil), indicating the desorption of Cd induced by direct contact of bacteria with soil particles. The median bioavailable fraction of Cd (1%) was 23-fold lower than the fraction extracted by acetic acid. The Pb-Cd sensor Staphylococcus aureus (pT0024) detected bioavailable Pb only in the suspensions of five of the most lead-polluted soils (>417 mg Pb/kg): the median bioavailability of Pb was 0.42%. Consequently, the hazard assessment relying on total metal levels in soils should be revised by critical comparison with data obtained from bioassays. Development and use of biosensors (excellent tools for mechanistic studies and signaling hazard already at subtoxic level) should be encouraged.

A novel method for comparison of biocidal properties of nanomaterials to bacteria, yeasts and algae.

Toxicity testing of nanomaterials (NMs) is experimentally challenging because NMs may interfere with test environment and assay components. In this work we propose a simple and reliable method--a 'spot test' to compare biocidal potency of NMs to unicellular microorganisms such as bacteria, yeasts and algae. The assay is straightforward: cells are incubated in deionized water suspensions of NMs for up to 24h and then pipetted as a 'spot' on agarized medium. Altogether seven bacterial strains, yeast and a microalga were tested. CuO, TiO2 and two different Ag NPs, multi-wall C-nanotubes (MWCNTs), AgNO3, CuSO4, 3,5-dichlorophenol, triclosan and H2O2 were analyzed. The biocidal potency of tested substances ranged from 0.1mg/L to >1000 mg/L; whereas, the least potent NMs toward all test species were TiO2 NPs and MWCNTs and most potent Ag and CuO NPs. Based on the similar toxicity pattern of the tested chemicals on the nine unicellular organisms in deionized water we conclude that toxicity mechanism of biocidal chemicals seems to be similar, whatever the organism (bacteria, yeast, alga). Therefore, when the organisms are not 'protected' by their environment that usually includes various organic and inorganic supplements their tolerance to toxicants is chemical- rather than organism-dependent.

Size-dependent toxicity of silver nanoparticles to bacteria, yeast, algae, crustaceans and mammalian cells in vitro.

The concept of nanotechnologies is based on size-dependent properties of particles in the 1-100 nm range. However, the relation between the particle size and biological effects is still unclear. The aim of the current paper was to generate and analyse a homogenous set of experimental toxicity data on Ag nanoparticles (Ag NPs) of similar coating (citrate) but of 5 different primary sizes (10, 20, 40, 60 and 80 nm) to different types of organisms/cells commonly used in toxicity assays: bacterial, yeast and algal cells, crustaceans and mammalian cells in vitro. When possible, the assays were conducted in ultrapure water to minimise the effect of medium components on silver speciation. The toxic effects of NPs to different organisms varied about two orders of magnitude, being the lowest (∼0.1 mg Ag/L) for crustaceans and algae and the highest (∼26 mg Ag/L) for mammalian cells. To quantify the role of Ag ions in the toxicity of Ag NPs, we normalized the EC50 values to Ag ions that dissolved from the NPs. The analysis showed that the toxicity of 20-80 nm Ag NPs could fully be explained by released Ag ions whereas 10 nm Ag NPs proved more toxic than predicted. Using E. coli Ag-biosensor, we demonstrated that 10 nm Ag NPs were more bioavailable to E. coli than silver salt (AgNO3). Thus, one may infer that 10 nm Ag NPs had more efficient cell-particle contact resulting in higher intracellular bioavailability of silver than in case of bigger NPs. Although the latter conclusion is initially based on one test organism, it may lead to an explanation for "size-dependent" biological effects of silver NPs. This study, for the first time, investigated the size-dependent toxic effects of a well-characterized library of Ag NPs to several microbial species, protozoans, algae, crustaceans and mammalian cells in vitro.

Particle-cell contact enhances antibacterial activity of silver nanoparticles.

BACKGROUND: It is generally accepted that antibacterial properties of Ag nanoparticles (AgNPs) are dictated by their dissolved fraction. However, dissolution-based concept alone does not fully explain the toxic potency of nanoparticulate silver compared to silver ions. METHODOLOGY/PRINCIPAL FINDINGS: Herein, we demonstrated that the direct contact between bacterial cell and AgNPs' surface enhanced the toxicity of nanosilver. More specifically, cell-NP contact increased the cellular uptake of particle-associated Ag ions - the single and ultimate cause of toxicity. To prove that, we evaluated the toxicity of three different AgNPs (uncoated, PVP-coated and protein-coated) to six bacterial strains: Gram-negative Escherichia coli, Pseudomonas fluorescens, P. putida and P. aeruginosa and Gram-positive Bacillus subtilis and Staphylococcus aureus. While the toxicity of AgNO3 to these bacteria varied only slightly (the 4-h EC50 ranged from 0.3 to 1.2 mg Ag/l), the 4-h EC50 values of protein-coated AgNPs for various bacterial strains differed remarkably, from 0.35 to 46 mg Ag/l. By systematically comparing the intracellular and extracellular free Ag(+) liberated from AgNPs, we demonstrated that not only extracellular dissolution in the bacterial test environment but also additional dissolution taking place at the particle-cell interface played an essential role in antibacterial action of AgNPs. The role of the NP-cell contact in dictating the antibacterial activity of Ag-NPs was additionally proven by the following observations: (i) separation of bacterial cells from AgNPs by particle-impermeable membrane (cut-off 20 kDa, ∼4 nm) significantly reduced the toxicity of AgNPs and (ii) P. aeruginosa cells which tended to attach onto AgNPs, exhibited the highest sensitivity to all forms of nanoparticulate Ag. CONCLUSIONS/SIGNIFICANCE: Our findings provide new insights into the mode of antibacterial action of nanosilver and explain some discrepancies in this field, showing that "Ag-ion" and "particle-specific" mechanisms are not controversial but, rather, are two faces of the same coin.

Interactions of PLA2-s from Vipera lebetina, Vipera berus berus and Naja naja oxiana venom with platelets, bacterial and cancer cells.

Secretory phospholipasesA(2) (sPLA(2)s) form a large family of structurally related enzymes widespread in nature. Herein, we studied the inhibitory effects of sPLA(2)s from Vipera lebetina (VLPLA(2)), Vipera berus berus (VBBPLA(2)), and Naja naja oxiana (NNOPLA(2)) venoms on (i) human platelets, (ii) four different bacterial strains (gram-negative Escherichia coli and Vibrio fischeri; gram-positive Staphylococcus aureus and Bacillus subtilis) and (iii) five types of cancer cells (PC-3, LNCaP, MCF-7, K-562 and B16-F10) in vitro. sPLA(2)s inhibited collagen-induced platelet aggregation: VBBPLA(2) IC(50) = 0.054, VLPLA(2) IC(50) = 0.072, NNOPLA(2) IC(50) = 0.814 µM. p-Bromophenacylbromide-inhibited sPLA(2) had no inhibitory action on platelets. 36.17 µM VBBPLA(2 )completely inhibited the growth of gram-positive Bacillus subtilis whereas no growth inhibition was observed towards gram-negative Escherichia coli. The inhibitory action of sPLA(2)s (~0.7 µM and ~7 µM) towards cancer cells depended on both venom and cell type. VBBPLA(2 )(7.2 µM) inhibited significantly the viability of K-562 cells and the cell death appeared apoptotic. The sPLA(2)s exhibited no inhibitory effect towards LNCaP cells and some effect (8%-20%) towards other cells. Thus, already sub-µM concentrations of sPLA(2)s inhibited collagen-induced platelet aggregation and from the current suite of studied svPLA(2)s and test cells, VBBPLA(2) was the most growth inhibitory towards Bacillus subtilis and K-562 cells.

Environmental hazard of oil shale combustion fly ash.

The combined chemical and ecotoxicological characterization of oil shale combustion fly ash was performed. Ash was sampled from the most distant point of the ash-separation systems of the Balti and Eesti Thermal Power Plants in North-Eastern Estonia. The fly ash proved potentially hazardous for tested aquatic organisms and high alkalinity of the leachates (pH>10) is apparently the key factor determining its toxicity. The leachates were not genotoxic in the Ames assay. Also, the analysis showed that despite long-term intensive oil-shale combustion accompanied by considerable fly ash emissions has not led to significant soil contamination by hazardous trace elements in North-Eastern Estonia. Comparative study of the fly ash originating from the 'new' circulating fluidized bed (CFB) combustion technology and the 'old' pulverized-fired (PF) one showed that CFB fly ash was less toxic than PF fly ash. Thus, complete transfer to the 'new' technology will reduce (i) atmospheric emission of hazardous trace elements and (ii) fly ash toxicity to aquatic organisms as compared with the 'old' technology.

Toxicity testing of heavy-metal-polluted soils with algae Selenastrum capricornutum: a soil suspension assay.

A small-scale Selenastrum capricornutum (Rhapidocelis subcapitata) growth inhibition assay was applied to the toxicity testing of suspensions of heavy-metal-polluted soils. The OECD 201 standard test procedure was followed, and algal biomass was measured by the fluorescence of extracted chlorophyll. The soils, which contained up to (per kilogram) 1390 mg of Zn, 20 mg of Cd, and 1050 mg of Pb were sampled around lead and zinc smelters in northern France. The water extractability of the metals in suspensions (1 part soil/99 parts water w/v) was not proportional to the pollution level, as extractability was lower for soil samples that were more polluted. Thus, the same amount of metals could be leached out of soils of different levels of pollution, showing that total concentrations of heavy metals in soil (currently used for risk assessment purposes) are poor predictors of the real environmental risk via the soil-water path. Despite high concentrations of water-extracted zinc (0.6-1.4 mg/L of Zn in the test), exceeding by approximately 10-fold the EC(50) value for S. capricornutum (0.1 mg Zn/L), 72-h algal growth in the soil extracts was comparable or better than growth in the standard control OECD mineral medium. The soil suspension stimulated the growth of algae up to eightfold greater than growth using the OECD control medium. Growth stimulation of algae was observed even when soil suspensions contained up to 12.5 mg Zn/L and could not be explained by supplementary nitrogen, phosphorous, and carbonate leached from the soil. However, if the growth of algae in suspensions of clean and polluted soils was compared, a dose-dependent inhibitory effect of metals on algal growth was demonstrated. Thus, as soil contains nutrients/supplements that mask the adverse effect of heavy metals, a clean soil that has properties similar to the polluted soils should be used instead of mineral salt solution as a control for analysis of the ecotoxicity of soils.