Abstract This article reports novel results on the toxic mechanisms of action of amine- and hydroxyl-terminated poly(amidoamine) (PAMAM) dendrimers toward microorganisms of environmental relevance, namely a cyanobacterium of the genus Anabaena and the green alga Chlamydomonas reinhardtii. We used PAMAM ethylenediamine core dendrimers from generations G2 to G4, which displayed a positive charge, measured as ?-potential, in culture media. All amine-terminated and most remarkably the G4 hydroxyl-terminated dendrimer inhibited the growth of both microorganisms. The effect on the growth of the green alga was significantly higher than that on the cyanobacterium. With concentrations expressed in terms of molarity, there was a clear relationship between dendrimer generation and toxicity, with higher toxicity for higher generation. Hormesis was observed for hydroxyl-terminated dendrimers at low concentrations. The cationic dendrimers and G4-OH significantly increased the formation of reactive oxygen species (ROS) in both organisms. ROS formation was not related with the chloroplast or photosynthetic membranes and photosystem II photochemistry was unaffected. Cell damage resulted in cytoplasm disorganization and cell deformities and was associated to an increase in ROS formation and lipid peroxidation in mitochondria in the green alga; cell wall and membrane disruption with apparent loss of cytoplasmic contents was found in the cyanobacterium. It was determined for the first time that cationic PAMAM dendrimers were quickly and largely internalized by both organisms. These results warn against the generalization of the use of dendrimers, which may pose significant risk for the environment and particularly for primary producers which are determinant for the health of natural ecosystems.
This chapter deals with the use of bioluminescent microorganisms in environmental monitoring, particularly in the assessment of the ecotoxicity of pollutants. Toxicity bioassays based on bioluminescent microorganisms are an interesting complement to classical toxicity assays, providing easiness of use, rapid response, mass production, and cost effectiveness. A description of the characteristics and main environmental applications in ecotoxicity testing of naturally bioluminescent microorganisms, covering bacteria and eukaryotes such as fungi and dinoglagellates, is reported in this chapter. The main features and applications of a wide variety of recombinant bioluminescent microorganisms, both prokaryotic and eukaryotic, are also summarized and critically considered. Quantitative structure-activity relationship models and hormesis are two important concepts in ecotoxicology; bioluminescent microorganisms have played a pivotal role in their development. As pollutants usually occur in complex mixtures in the environment, the use of both natural and recombinant bioluminescent microorganisms to assess mixture toxicity has been discussed. The main information has been summarized in tables, allowing quick consultation of the variety of luminescent organisms, bioluminescence gene systems, commercially available bioluminescent tests, environmental applications, and relevant references.
Aggregation raises attention in Nanotoxicology due to its methodological implications. Aggregation is a physical symptom of a more general physicochemical condition of colloidal particles, namely, colloidal stability. Colloidal stability is a global indicator of the tendency of a system to reduce its net surface energy, which may be achieved by homo-aggregation or hetero-aggregation, including location at bio-interfaces. However, the role of colloidal stability as a driver of ENM bioactivity has received little consideration thus far. In the present work, which focuses on the toxicity of nanoscaled Fe° nanoparticles (nZVI) towards a model microalga, we demonstrate that colloidal stability is a fundamental driver of ENM bioactivity, comprehensively accounting for otherwise inexplicable differential biological effects. The present work throws light on basic aspects of Nanotoxicology, and reveals a key factor which may reconcile contradictory results on the influence of aggregation in bioactivity of ENMs.
The individual and combined toxicities of amoxicillin, erythromycin, levofloxacin, norfloxacin and tetracycline have been examined in two organisms representative of the aquatic environment, the cyanobacterium Anabaena CPB4337 as a target organism and the green alga Pseudokirchneriella subcapitata as a non-target organism. The cyanobacterium was more sensitive than the green alga to the toxic effect of antibiotics. Erythromycin was highly toxic for both organisms; tetracycline was more toxic to the green algae whereas the quinolones levofloxacin and norfloxacin were more toxic to the cyanobacterium than to the green alga. Amoxicillin also displayed toxicity to the cyanobacterium but showed no toxicity to the green alga. The toxicological interactions of antibiotics in the whole range of effect levels either in binary or multicomponent mixtures were analyzed using the Combination Index (CI) method. In most cases, synergism clearly predominated both for the green alga and the cyanobacterium. The CI method was compared with the classical models of additivity Concentration Addition (CA) and Independent Action (IA) finding that CI could accurately predict deviations from additivity. Risk assessment was performed by calculating the ratio between Measured Environmental Concentration (MEC) and the Predicted No Effect Concentration (PNEC). A MEC/PNEC ratio higher than 1 was found for the binary erythromycin and tetracycline mixture in wastewater effluents, a combination which showed a strong synergism at low effect levels in both organisms. From the tested antibiotic mixtures, it can be concluded that certain specific combinations may pose a potential ecological risk for aquatic ecosystems with the present environmentally measured concentrations.
The combined toxicity of the perfluorinated surfactants perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA) and several pollutants (Hg(2+), Cd(2+), 2,4-D, propylparaben, mitomycin C and furazolidone) has been examined with a bioluminescent cyanobacterial toxicity test. Hg(2+), Cd(2+), mitomycin C and furazolidone could be included in the "Acute aquatic hazard" category established in the Regulation (EC) No 1272/2008 being "very toxic to aquatic life". Toxicological interactions of PFOA, PFOS with these pollutants in binary, ternary and multicomponent mixtures were studied using the combination-index method. PFOA and PFOS showed an antagonistic interaction at the whole range of effect levels, this may explain in part the finding that PFOA and PFOS interacted in an inverse way with the organic pollutants; the relative hydrophobicity of the tested compounds would also explain this interaction pattern. The interaction of both PFOS and PFOA with heavy metals was mostly antagonistic, decreasing metal toxicity. With increasing complexity of the mixtures, the CI method predicted synergism at low to very low levels of effect; pollutant combinations at their mixture NOECs were tested and confirmed the predicted synergism.
The physicochemical properties of nanoparticles determine their interaction with living organisms. Four different cerium oxide nanoparticles, including commercial materials, were characterized and compared with a micron-sized ceria. The formation of aggregates as well as ?-potential, surface area, and chemical composition were determined. The formation of primary particle aggregates was a slow process that led to different particle sizes depending on the composition of the medium. In this paper, we describe the toxicity of cerium oxide for the self-luminescent cyanobacterial recombinant strain Anabaena CPB4337 and the green alga Pseudokirchneriella subcapitata. The toxicity for Anabaena exposed to nanoparticles in pure water for 24 h ranged from 0.27 to 6.3 mg/l; P. subcapitata EC(50) (yielded effective concentration of nanoparticles that inhibits the cellular function of interest by 50%) values in the 2.4-29.6 mg/l range. Images of both organisms showed membrane disruption and highly damaged cells. Free cerium was highly toxic for both organisms, but the negligible amount found dissolved in the nanoparticle suspensions could not explain the observed toxic effect of nanoceria on the aquatic organisms; the dissolution of zinc could contribute to the toxicity of bulk material but could not explain the toxic effect of nanoceria either. We found no evidence of nanoparticle uptake by cells, but our observations suggested that their toxic mode of action required direct contact between nanoparticles and cells; in the case of the cyanobacterium, cells completely coated by layers of ceria nanoparticles were observed. Cell damage most probably took place by cell wall and membrane disruption; further research is needed to find out whether the oxidative activity of ceria could be responsible.
Calcium functions as a versatile messenger in a wide variety of eukaryotic and prokaryotic cells. Cyanobacteria are photoautotrophs which have a great ecological impact as primary producers. Our research group has presented solid evidence of a role of calcium in the perception of environmental changes by cyanobacteria and their acclimation to these changes. We constructed a recombinant strain of the freshwater cyanobacterium Anabaena sp. PCC 7120 that constitutively expresses the calcium-binding photoprotein apoaequorin, enabling in-vivo monitoring of any fluctuation in the intracellular free calcium concentration of the cyanobacterium in response to any environmental stimulus. The "Ca(2+) signature" is the combination of changes in all Ca(2+) signal properties (magnitude, duration, frequency, source of the signal) produced by a specific stimulus. We recorded and analyzed the Ca(2+) signatures generated by exposure of the cyanobacterium to different groups of environmental pollutants, for example cations, anions, organic solvents, naphthalene, and pharmaceuticals. We found that, in general, each group of tested chemicals triggered a specific calcium signature in a reproducible and dose-dependent manner. We hypothesize that these Ca(2+) signals may be related to the cellular mechanisms of pollutant perception and ultimately to their toxic mode of action. We recorded Ca(2+) signals triggered by binary mixtures of pollutants and a signal induced by a real wastewater sample which could be mimicked by mixing its main constituents. Because Ca(2+) signatures were induced before toxicity was evident, we propose that intracellular free Ca(2+) may serve as an early biomarker of exposure to environmental pollution.
The toxicity of perfluorinated surfactants perfluorooctane sulfonic acid (PFOS), perfluorooctanoic acid (PFOA), perfluorobutane sulfonate (PFBS) and PF-656 as well as the sulfosuccinate surfactant docusate sodium has been examined using two bioluminescence inhibition assays based on the marine bacterium Vibrio fischeri and the self-luminescent cyanobacterial recombinant strain Anabaena CPB4337. We also determined multigenerational toxicity towards the growth of the algae Pseudokirchneriella subcapitata. With EC(50) values in the 43-75 mg/L range, docusate sodium exhibited a higher toxicity towards the three organisms than PFOS, PFOA, PF-656 and PFBS. We investigated the toxicological interactions of the most toxic surfactant, docusate sodium, with two chlorinated compounds, triclosan and 2,4,6-trichlorophenol (TCP), in their binary and ternary mixtures using the method of the combination index based on the median-effect equation. In general, the binary mixture of the chlorinated compounds triclosan and TCP exhibited antagonism, which was stronger for the growth test using P. subcapitata. Except for the green alga, the binary mixtures of docusate sodium with TCP or triclosan showed synergism at medium to high effect levels; the synergistic behaviour predominating in the ternary mixture and in the three tested species. This result highlights the potential toxicological risk associated with the co-occurrence of this surfactant with other pollutants.
Pharmaceuticals in the aquatic environment do not appear singly and usually occur as complex mixtures, whose combined effect may exhibit toxicity to the aquatic biota. We report an environmental application of the combination index (CI)-isobologram equation, a method widely used in pharmacology to study drug interactions, to determine the nature of toxicological interactions of three fibrates toward two aquatic bioluminescent organisms, Vibrio fischeri and the self-luminescent cyanobacterial recombinant strain Anabaena CPB4337. The combination index-isobologram equation method allows computerized quantitation of synergism, additive effect and antagonism. In the Vibrio test, the fibrate combinations showed antagonism at low effect levels that turned into an additive effect or synergism at higher effect levels; by contrast, in the Anabaena test, the fibrate combinations showed a strong synergism at the lowest effect levels and a very strong antagonism at high effect levels. We also evaluated the nature of the interactions of the three fibrates with a real wastewater sample in the cyanobacterial test. We propose that the combination index-isobologram equation method can serve as a useful tool in ecotoxicological assessment.
The high consumption of blood lipid regulators is leading to frequent reports of the occurrence of fibrates in natural streams and wastewater effluents. This paper describes a study undertaken to evaluate the acute toxicity of bezafibrate, clofibric acid, gemfibrozil, and fenofibric acid, a metabolite of fenofibrate whose ecotoxicity has not been previously reported.
The bioavailability and therefore toxicity of a metal depends on the chemical species present in a particular environment. We evaluated the effect of a series of factors that could potentially modify metal speciation on the toxicity of Hg, Cu, Zn, and Cd toward a recombinant strain of the freshwater cyanobacterium Anabaena sp. PCC 7120 with cloned lux operon of luminescent terrestrial bacterium Photorhabdus luminescens. The strain, denoted as Anabaena CPB4337, showed a high constitutive luminescence with no need to add exogenous aldehyde. The tested factors were pH, EDTA (as organic ligand), and anions PO(4)(3-), CO(3)(2-), and Cl(-). Chemical modeling and correlation analyses were used to predict metal speciation and link it with toxicity. In general, metal toxicity significantly correlated to the predicted metal free-ion concentration, although Zn-EDTA complexes and certain Hg chloro-complexes could also exhibit some toxicity to cyanobacteria. An interesting feature of metal toxicity to strain Anabaena CPB4337 was that low amounts of PO(4)(3-) and CO(3)(2-) increased metal toxicity; this effect could not be related to significant changes in metal speciation and could be attributed to a modulating effect of these anions on metal/uptake toxicity. The combination of toxicity studies that take into account a range of factors that might modulate metal toxicity with chemical modeling to predict changes in metal speciation might be useful for interpreting complex toxicity data. Finally, this cyanobacterial bioreporter, due to its ecological relevance as a primary producer, could be used as a tool for toxicity assessment in freshwater environments.
The aim of this study was to validate the effectiveness of a phytoremediation procedure for metal-working fluids (MWFs) with maize plants growing in hydroponic culture in which the roots grow on esparto fibre and further improve bioremediation potential of the system with root beneficial bacteria, seeking a synergistic effect of the plant-microorganism combination. Chemical oxygen demand (COD), pH, total and type of hydrocarbons measured after phytoremediation indicated that the process with maize plants was successful, as demonstrated by the significant decrease in the parameters measured. This effect was mainly due to the plant although inoculated microorganisms had a relevant effect on the type of remaining hydrocarbons. The success of the phytoremediation process was further confirmed by two toxicity tests, one of them based on chlorophyll fluorescence measurements on maize plants and another one based on cyanobacteria, using a bioluminescent toxicity bioassay; both tests demonstrated that the phytoremediated waste was significantly less toxic than the initial non-phytoremediated MWFs.
The effect of nanoceria on two aquatic photosynthetic organisms of ecological relevance, a green alga and a cyanobacterium, is reported. The main bioenergetic process of these organisms, photosynthesis, was studied by measuring both oxygen evolution and chlorophyll a fluorescence emission parameters. Nanoceria significantly inhibited photosynthesis in the cyanobacterium in the entire range of concentrations tested (0.01-100 mg/L), while a dual effect of nanoceria was found in the green alga with slight stimulation at low concentrations and strong inhibition at the highest concentrations tested. Chlorophyll a fluorescence experiments indicated that nanoceria had a significant impact on the primary photochemical processes of photosystem II. The primary cause of the observed photosynthetic inhibition by nanoceria is an excessive level of ROS formation; the results indicated a strong generation of reactive oxygen species (ROS) which caused oxidative damage, as evidenced by lipid peroxidation in both photosynthetic organisms. It is proposed that nanoceria can increase the production of hydrogen peroxide (a normal ROS by-product of light-driven photosynthesis) in both the green alga and the cyanobacterium; through an oxidative reaction, these ROS cause lipid peroxidation, compromising membrane integrity and also seriously impairing photosynthetic performance, eventually leading to cell death.
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