Toxic effects of nanoparticles on bioluminescence activity,seed germination,and gene mutation

The potential environmental toxicities of several metal oxide nanoparticles (NPs; CuO, TiO₂, NiO, Fe₂O₃, ZnO, and Co₃O₄) were evaluated in the context of bioluminescence activity, seed germination, and bacterial gene mutation. The bioassays exhibited different sensitivities, i.e., each kind of NP exhibited a different level of toxicity in each of the bioassays. However, with a few exceptions, CuO and ZnO NPs had most toxic for germination of Lactuca seed (EC₅₀ 0.46 mg CuO/l) and bioluminescence (EC₅₀ 1.05 mg ZnO/l). Three NPs (Co₃O₄, TiO₂, and Fe₂O₃) among all tested concentrations (max. 1,000 mg/l) showed no inhibitory effects on the tested organisms, except for Co₃O₄ NPs on bioluminescence activity (EC₅₀ 62.04 mg/l). The sensitivity of Lactuca seeds was greater than that of Raphanus seeds (EC₅₀ 0.46 mg CuO/l versus 26.84 mg CuO /l ). The ranking of metal toxicity levels on bioluminescence was in the order of ZnO?>?CuO?>?Co₃O₄?>?NiO?>?Fe₂O₃, TiO₂, while CuO?>?ZnO?>?NiO?>?Co₃O₄, Fe₂O₃, TiO₂ on germination. No revertant mutagenic ratio (greater than 2.0) of Salmonella typhimurium TA 98 was observed under any tested condition. These findings demonstrate that several bioassays, as opposed to any single one, are needed for the accurate assessment of NP toxicity on ecosystems.     

Assessment of Arsenite,Arsenate, and Chromate Phytotoxicity Based on the Activity of Seed Germination and Growth (Root & Shoot) of Various Plant Seeds

Effects of different concentrations of arsenite, arsenate, and chromate on seed germination, root length (RL), and shoot length (SL) in four seed types, chosen from preliminary tests with eight seed types, were investigated to assess the toxicity of the tested metals. The sensitivities of the four different seed types toward germination, relative RL (RRL), and relative SL (RSL) varied with each metal. In a comparison, the germination of the seeds was more sensitive to the tested metals than the other chosen endpoints (RL and SL). Arsenite was generally more restrictive to all the endpoints (germination, root, and shoot growth) than arsenate and chromate. Lactuca (garden lettuce) was also generally more sensitive to the tested metals than the other seed types. The correlation between RRL and RSL varied depending on the seed type and metal tested. However, significant correlations (r² > 0.85) of these were observed with Lactuca seeds, which appeared to be an optimal plant with respect to the tolerance of the tested metals. The differences in the toxicities of metals toward different plant species should be taken into account in the bioassessment of metals-contaminated sites. Thus, this study encourages the need to combine the three endpoints of various seeds in the evaluation of toxicities of metals.     

The Effect of Arsenate and Other Inhibitors on Early Events during the Germination of Lettuce Seeds (Lactuca sativa L.)

The effect of arsenate, arsenite, 2,4-dinitrophenol, and anaerobiosis on early events in seed germination was investigated using both intact and punched seeds of lettuce (Lactuca sativa L.). It was found that punching the seed removes penetration barriers to the entrance of inhibitors without an undue loss of germination or light responses. The kinetics of the action of germination inhibitors were established by 2-hour pulse experiments. Arsenate and 2, 4-dinitrophenol have very different kinetics. The inhibition of germination in punched seeds by arsenate given in conjunction with phosphate compared with the lack of inhibition of arsenate plus phosphate on the growing seedling, suggest a distinct metabolic change in the germinating embryo at some time between the onset of germination and subsequent seedling growth.     

Relative toxicity of arsenite and arsenate on germination and early seedling growth of rice (Oryza sativa L.)

Elevated soil arsenic levels resulting from long-term use of arsenic contaminated ground for irrigation in Bangladesh may inhibit seed germination and seedling establishment of rice, the country's main food crop. A germination study on rice seeds and a short-term toxicity experiment with different concentrations of arsenite and arsenate on rice seedlings were conducted. Percent germination over control decreased significantly with increasing concentrations of arsenite and arsenate. Arsenite was found to be more toxic than arsenate for rice seed germination. There were varietal differences among the test varieties in response to arsenite and arsenate exposure. The performance of the dry season variety Purbachi was the best among the varieties. Germination of Purbachi was not inhibited at all up to 4 mg l^–1 arsenite and 8 mg l^–1 arsenate treatment. Root tolerance index (RTI) and relative shoot height (RSH) for rice seedlings decreased with increasing concentrations of arsenite and arsenate. Reduction of RTI caused by arsenate was higher than that of arsenite. In general, dry season varieties have more tolerance to arsenite or arsenate than the wet season varieties.     

Improved germination bioassays for allelopathy research

A new method for germination bioassays in allelopathy was evaluated. Inspired by pharmacology, allelochemical quantity to test on different target seeds was calculated according to seeds biometry. The first step consisted in measuring volume, mass, surface and shape of Lactuca sativa and Raphanus sativus seeds. The radish seed mass, surface, volume and contact area biometric parameters were respectively 10, 2, 6 and 1.7 that of lettuce. Two germination bioassay sets were compared: (i) a conventional one, testing the same concentration of allelochemical (2-benzoxazolinone termed as “BOA”) on the two species and, (ii) a biometrics enhanced seed test (“BEST” method), employing quantities of the chemical that were calculated in proportion to seed biometry parameters. The conventional method indicated that 1 mM BOA slowed and decreased germination rate (radish 50 %, lettuce 10 %) whereas 10 and 0.1 μM did not induce any effect. The BEST method offered more differentiated results: applied BOA according to seed volume induced significant inhibition of radish germination (both quantities), while lettuce was only affected by the highest dose. The same occurred when considering grain surface and contact area. Thus, the BEST assay showed more clearly that radish was more sensitive to BOA than lettuce. Compared to a setup with identical test compound concentrations for all tested species, the BEST method provides more differentiated results contributing to a more realistic comparative susceptibility assessment.     

Effects of Temperature and Various Red or Far-red Irradiations on Phytochrome- and Gibberellin A3-mediated Germination Control in Partially Hydrated Lettuce Seeds

Dark reversion of phytochrome in partially hydrated lettuceseeds (Lactuca sativa cv. Grand Rapids) is temperature dependent.After initial red irradiation (R) the higher the storage temperature,the higher the dark reversion rate. Following dark moist storage(DMS) at 30 ?C for 15 d none of the seeds receiving initialR germinated, whereas seeds stored at 0 ?C germinated nearlyas well (about 80%) as unstored controls. The half-time fordark reversion at 20 ?C and 30 ?C is 9 d and 3 d respectively.Repeated R treatments given at 5 d intervals during DMS at 20?C and 30 ?C maintained a high germination capacity. With threeor more R treatments the effect of high temperature largelydisappeared. Dark reversion of phytochrome was not observed in partiallyhydrated lettuce seeds receiving continuous red irradiation(cont R) for two or more days. The promotive effect of contR could be reversed at any time with a brief far-red irradiation(FR), indicating that the phytochrome system remained fullyphototransformable. With continuous far-red light (cont FR)the ability of gibberellin A₃ (GA₃) to stimulate germinationdisappeared and response to GA₃ also diminished in cont R followedby FR but at a slower rate indicating the induction of secondarydormancy in these partially hydrated seeds. This induction ofdormancy was retarded by repetitive or cont R but was enhancedby cont FR. The results of this study suggest a role for theaccumulated stable intermediates of phytochrome transformationin partially hydrated seeds with repeated or continuous R treatmentsand different effects of GA₃ and R in the regulation of germination. Key words: Phytochrome, Lactuca sativa, Seed germination, Temperature, Dark reversion of phytochrome, Seed water content     

Inducible plasmid-determined resistance to arsenate, arsenite, and antimony (III) in escherichia coli and Staphylococcus aureus.

Plasmids in both Escherichia coli and Staphylococcus aureus contain an "operon" that confers resistance to arsenate, arsenite, and antimony(III) salts. The systems were always inducible. All three salts, arsenate, arsenite, and antimony(III), were inducers. Mutants and a cloned deoxyribonucleic acid fragment from plasmid pI258 in S. aureus have lost arsenate resistance but retained resistances to arsenite and antimony, demonstrating that separate genes are involved. Arsenate-resistant arsenite-sensitive S. aureus plasmid mutants were also isolated. In E. coli, plasmid-determined arsenate resistance and reduced uptake were additive to that found with chromosomal arsenate resistance mutants. Arsenate resistance was due to reduced uptake of arsenate by the induced plasmid-containing cells. Under conditions of high arsenate, when some uptake could be demonstrated with the induced resistant cells, the arsenate was rapidly lost by the cells in the absence of extracellular phosphate. Sensitive cells retained arsenate under these conditions. When phosphate was added, phosphate-arsenate exchange occurred. High phosphate in the growth medium protected cells from arsenate, but not from arsenite or antimony(III) toxicity. We do not know the mechanisms of arsenite or antimony resistance. However, arsenite was not oxidized to less toxic arsenate. Since cell-free medium "conditioned" by prior growth to induced resistant cells with toxic levels of arsenite or antimony(III) retained the ability to inhibit the growth of sensitive cells, the mechanism of arsenite and antimony resistance does not involve conversion of AsO2- or SbO+ to less toxic forms or binding by soluble thiols excreted by resistant cells.     

Comparative investigations of sodium arsenite, arsenic trioxide and cadmium sulphate in combination with gamma-radiation on apoptosis, micronuclei induction and DNA damage in a human lymphoblastoid cell line

In the field of radiation protection the combined exposure to radiation and other toxic agents is recognised as an important research area. To elucidate the basic mechanisms of simultaneous exposure, the interaction of the carcinogens and environmental toxicants cadmium and two arsenic compounds, arsenite and arsenic trioxide, in combination with gamma-radiation in human lymphoblastoid cells (TK6) were investigated. Gamma-radiation induced significant genotoxic effects such as micronuclei formation, DNA damage and apoptosis, whereas arsenic and cadmium had no significant effect on these indicators of cellular damage at non-toxic concentrations. However, in combination with gamma-radiation arsenic trioxide induced a more than additive apoptotic rate compared to the sum of the single effects. Here, the level of apoptotic cells was increased, in a dose-dependent way, up to two-fold compared to the irradiated control cells. Arsenite did not induce a significant additive effect at any of the concentrations or radiation doses tested. On the other hand, arsenic trioxide was less effective than arsenite in the induction of DNA protein cross-links. These data indicate that the two arsenic compounds interact through different pathways in the cell. Cadmium sulphate, like arsenite, had no significant effect on apoptosis in combination with gamma-radiation at low concentrations and, at high concentrations, even reduced the radiation-induced apoptosis. An additive effect on micronuclei induction was observed with 1 μM cadmium sulphate with an increase of up to 80% compared to the irradiated control cells. Toxic concentrations of cadmium and arsenic trioxide seemed to reduce micronuclei induction.

The results presented here indicate that relatively low concentrations of arsenic and cadmium, close to those occuring in nature, may interfere with radiation effects. Differences in action of the two arsenic compounds were identified.     

Phenolic Growth Inhibitors Isolated from Dormant Buds of Sugar Maple (Acer saccharum Marsh)

Phenolic growth-inhibiting substances were isolated from dormantbuds of sugar maple (Acer saccharurn Marsh) by paper chromatographyof their aqueous methanolic extracts. Inhibition was determinedin germination bioassays using lettuce (Lactuca sativa var.Grand Rapids) seeds. An inhibition of some 20% was attributedto four major phenolics identified as ferulic, vanillic, p-coumaric,and caffeic acids. Quercetm was also present and caused a weakstimulation of growth. Fractionation of the extract with acidic, neutral, and basicgroups indicated that more phenolic compounds were in the acidicgroup than in the neutral, while none were in the basic group. On a seasonal basis, autumn buds had a higher phenolic contentthan winter and spring buds. The results presented are not inconsistentwith the view that growth-inhibitory phenolic compounds couldcontribute to dormancy in sugar maple buds.     

Genetic variation for lettuce seed thermoinhibition is associated with temperature-sensitive expression of abscisic Acid, gibberellin, and ethylene biosynthesis, metabolism, and response genes

Lettuce (Lactuca sativa ‘Salinas’) seeds fail to germinate when imbibed at temperatures above 25°C to 30°C (termed thermoinhibition). However, seeds of an accession of Lactuca serriola (UC96US23) do not exhibit thermoinhibition up to 37°C in the light. Comparative genetics, physiology, and gene expression were analyzed in these genotypes to determine the mechanisms governing the regulation of seed germination by temperature. Germination of the two genotypes was differentially sensitive to abscisic acid (ABA) and gibberellin (GA) at elevated temperatures. Quantitative trait loci associated with these phenotypes colocated with a major quantitative trait locus (Htg6.1) from UC96US23 conferring germination thermotolerance. ABA contents were elevated in Salinas seeds that exhibited thermoinhibition, consistent with the ability of fluridone (an ABA biosynthesis inhibitor) to improve germination at high temperatures. Expression of many genes involved in ABA, GA, and ethylene biosynthesis, metabolism, and response was differentially affected by high temperature and light in the two genotypes. In general, ABA-related genes were more highly expressed when germination was inhibited, and GA- and ethylene-related genes were more highly expressed when germination was permitted. In particular, LsNCED4, a gene encoding an enzyme in the ABA biosynthetic pathway, was up-regulated by high temperature only in Salinas seeds and also colocated with Htg6.1. The temperature sensitivity of expression of LsNCED4 may determine the upper temperature limit for lettuce seed germination and may indirectly influence other regulatory pathways via interconnected effects of increased ABA biosynthesis.     

An examination of the growth substance activity of vitamin D3

The plant growth activity of vitamin D₃ has been examined using well-established bioassays. Vitamin D₃ was found to promote adventitious root formation in cuttings ofPopulus tremula and to promote the germination of lettuce (Lactuca sativa cv. Grand Rapids) seeds in the absence of light. No other effects were observed typical of plant growth substances.Presented at the Federation of European Societies of Plant Physiology IVth Congress, Strasbourg (1984).     

Short-term toxic effects of naphthalene and pyrene on the common prawn (Palaemon serratus) assessed by a multi-parameter laboratorial approach: mechanisms of toxicity and impairment of individual fitness

The short-term (96?h) toxic effects of two polycyclic aromatic hydrocarbons (PAHs), naphthalene (NAP) and pyrene (PYR), on the common prawn (Palaemon serratus) were investigated in laboratory bioassays, including a fitness related assay based on the post-exposure swimming velocity. Other effect criteria were biomarkers of neurotoxicity, oxidative stress and bioenergetics, and mortality. In the range of concentrations tested (NAP: 0.13–8?mg/L; PYR: 0.006–0.4?mg/L), both PAHs impaired the swimming velocity, induced oxidative stress and damage, and changed the activity of lactate dehydrogenase and isocitrate dehydrogenase. NAP also caused mortality (96 h-LC50?=?3.5?mg/L). Thus, both PAHs were able to cause toxic effects on P. serratus after a short period of exposure through the water, including the reduction of individual fitness. PYR was five folds more effective in reducing the swimming velocity of P. serratus than NAP. These findings are of interest for the marine ecological risk assessment of oil spills.     

Arsenite induced sensitization and self-tolerance of Reuber H35 hepatoma cells

Our data show that a short incubation with arsenite (30–300 M) induces a biphasic change in ceSlular sensitivity towards a second exposure to arsenite. A transient sensitization was followed by the development of self-tolerance. Sensitization was measured using the step-down protocol; i.e., application of a high dose of arsenite pretreatment (100 or 300 M) followed immediately by incubation in a low dose of arsenite (1–30 M), with extensive rinsing in between. Whereas no effect of 1 and 3 M on cellular survival is observed without pretreatment, a large decrease in cell survival can be established when these low doses of arsenite are applied immediately after a 1 hr pretreatment with 100 or 300 arsenite.According to the step-down protocol, a high dose of toxic compounds is applied and is followed by prolonged incubation in a lower concentration of the initial toxic compound. This might be a more accurate model for studying the effects of toxic insults on cells and organisms in the manner in which they occur in their natural environment. The level of tolerance was determined by a 1 hr test treatment with 300 pM arsenite applied at different times after pretreatment. Using this fractionated treatment protocol, it was established that tolerance increases with the increasing time intervals between the sodium arsenite treatments, during the 6 hr studied.These observations suggest that sensitization gradually decreases, whereas tolerance develops. Furthermore, our data indicate that the condition of pretreatment determines the extent to which the early sensitivity increases, as well as the development of tolerance later on. A relatively high arsenite concentration leads to more sensitized cells, which are transformed into more tolerant cells in comparison with the effect of a lower arsenite concentration.     

Genotoxic effects of potassium dichromate, sodium arsenite, cobalt chloride and lead nitrate in diploid yeast

4 metal salts, potassium dichromate, sodium arsenite, cobalt chloride and lead nitrate were tested for their genotoxic effects in Saccharomyces cerevisiae. Potassium dichromate was the most potent agent for induction of gene conversion and reverse mutation. Sodium arsenite was virtually ineffective as a convertogen but gave a positive result for reversion. Cobalt chloride was the least toxic, exhibited a convertogenic activity but was only marginally active for reverse mutation. Lead nitrate was the most toxic salt but was genetically inactive.     

Development of novel conductometric biosensors based on immobilised whole cell Chlorella vulgaris microalgae

A novel biosensor based on immobilised whole cell Chlorella vulgaris microalgae as a bioreceptor and interdigitated conductometric electrodes as a transducer has been developed and tested for alkaline phosphatase activity (APA) analysis. These sensors were also used for the detection of toxic compounds, namely cadmium ions, in aquatic habitats. Algae were immobilised inside bovine serum albumin (BSA) membranes cross-linked with glutaraldehyde vapours. The detection of the local conductivity variations caused by algae enzymatic reactions could be achieved. The inhibition of C. vulgaris microalgae Alkaline phosphatase activities in presence of cadmium ions was measured. These results were compared with measurements in bioassays. It finally appeared that conductometric biosensors using algae seemed more sensitive than bioassays to detect low levels of cadmium ions (the detection limit for the first experiments was 1 ppb of Cd2+). The main advantages of these alkaline phosphatase biosensors consist of their high specificity in regard to the toxic compounds they enable to detect, but also on their high stability since contrary to enzymatic biosensors, they use whole algae cells with APs on their walls.     

Hydroxamic acids in Secale cereale L. and the relationship with their antifeedant and allelopathic properties

Contents of the hydroxamic acids 2,4-dihydroxy-1,4-benzoxazin-3-one (DIBOA), and 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA) in leaves and roots of 14 cultivars of rye, Secale cereale L., were determined. Dynamics of accumulation in three cultivars were evaluated. DIBOA was the main cyclic hydroxamic acid in leaves but the contents differed significantly between the cultivars. Both DIBOA and DIMBOA were present in the roots. Maximum concentration of DIBOA in leaves and DIMBOA in roots was reached between 48-54 h and 54-72 h after germination, respectively. Antifeedant activity of DIBOA towards the aphid Rhopalosiphum padi and the feeding behavior were studied by electronic recording in barley leaves treated with different contents of DIBOA. The deleterious activity of DIBOA could arise by starvation and/or a toxic effect. Additionally, allelopathic potential of pure DIBOA and aqueous extracts of leaves and roots of rye (Tetra-Baer) on the germination of lettuce (Lactuca sativa) and rye (Tetra-Baer) seeds was evaluated. A high percentage of germination inhibition of pure DIBOA and the extracts of leaves and roots was observed. The activity is in agreement with the contents of hydroxamic acids in the plants. The substrates had no allelopathic effect on rye seeds.     

Assessment of the genotoxicity of contaminated soil with the Allium/Vicia-micronucleus and the Tradescantia-micronucleus assays.

The present study concerns the genotoxicity of contaminated soil near Metz, France. Three plant bioassays, the Vicia faba (broad bean), the Allium cepa (white onion) and the Tradescantia (spiderwort) micronuclei tests were used to evaluate for genotoxicity. Two soil samples were tested: soil sample A (from an industrial waste site) and soil sample B (from a cokeworks waste site). Maleic hydrazide was used as the positive control. Aqueous extracts of the soil samples were used to treat the roots of Vicia and Allium, and plant cuttings of Tradescantia according to the standard protocol for these plant assays established by the International Program on Chemical Safety and the World Health Organization. The results of these tests showed differential sensitivity in the three different bioassays. Soil sample A was more toxic than soil sample B.     

Insecticidal activity of Bacillus thuringiensis crystal proteins

Published data on insecticidal activity of crystal proteins from Bacillus thuringiensis are incorporated into the Bt toxin specificity relational database. To date, 125 of the 174 holotype known toxins have been tested in ∼1700 bioassays against 163 test species; 49 toxins have not been tested at all; 59 were tested against 71 Lepidoptera species in 1182 bioassays; 53 toxins were tested against 23 Diptera species in 233 bioassays; and 47 were tested against 39 Coleoptera species in 190 bioassays. Activity spectra of the tested toxins were summarized for each order. Comparisons of LC₅₀ values are confounded by high variability of the estimates, mostly due to within-species variation in susceptibility, and errors associated with estimation of toxin protein content. Limited analyses suggest that crystal protein toxicity is not affected by quarternary toxin rank or host used for gene expression, but that pre-ingestion treatment by solubilization or enzymatic processing has a large effect. There is an increasing number of toxin families with cross-order activity, as 15 of the 87 families (secondary rank) that are pesticidal are active against more than one order. Cross-order activity does not threaten environmental safety of B. thuringiensis-based pest control because toxins tend to be much less toxic to taxa outside the family’s primary specificity range.     

Arsenite-BAL as an Inhibitor of Germination

Imbibition of light-sensitive lettuce seeds in mixed solutions of sodium arsenite and 2,3-dimercaptopropanol (BAL) inhibits light-induced germination more potently than does arsenite alone, particularly at higher temperatures. There is limited reversal of the enhanced inhibition at high BAL/arsenite ratios. The complex is also more inhibitory than arsenite after shorter periods of imbibition. Monothiol compounds do not give the increased inhibition, nor do combinations of BAL with other thiol group reagents. Greater penetrability, possibly into some internal compartment of the cell, is suggested as the cause of the enhancement. Induction of germination by gibberellic acid or low temperature is equally sensitive to arsenite-BAL. The extent of the inhibition of germination of 9 other species by arsenite-BAL is not clearly related to their light-sensitivity.     

Algal bioassays to determine toxicity of metal mixtures

The shortcomings of many established toxicity criteria for metals have resulted from a simplification of bioassays performed with single metals. A more comprehensive approach was needed to diagnose the effects of metal mixtures on aquatic organisms. Using Chlorella, Ankistrodesmus and Scenedesmus as test organisms, we experimented on a number of factors which affected metal toxicity bioassays. These included metal interactions, algal competitions, species sensitivities, the ratio of an excess metal to other metals and nutrient levels. An alternative technique was finally established which involved an evaluation of individual assays of Chlorella and Ankistrodesmus in separate media and a tissue-metal analysis on Chlorella. Chlorella fusca, commonly found in lakes with high metal concentrations, showed high tolerance to mixed-metal solutions, while Ankistrodesmus proved to be very sensitive. By determining the maximum yield ratio between Ankistrodesmus and Chlorella (i.e. the A/Ch ratio) it was possible to compare the toxic strength of harmful metals according to an established standard curve of A/Ch ratio versus mixed-metal concentrations. The levels of tissue-metal analysed in Chlorella also gave some indication as to which metals were responsible for the toxicity.