Development of an in vitro batch-type closed gas exposure device with an alveolar epithelial cell line, A549, for toxicity evaluations of gaseous compounds.

To simply evaluate toxicity for various types of exhaust-gas samples collected in various locations, we developed a small-scale (150 mL) batch-type completely closed gas exposure device incorporated with an air-liquid interface culture of a human alveolar epithelial cell line, A549. On the basis of cell viability tests using an acid phosphatase assay after 48 h of gas exposure, the developed device was able to measure clear dose-response relationships for volatile organic and inorganic compounds, such as benzene, trichloroethylene (TCE), acetone, SO(2) and NO(2) gases, but not CO gas. Although the 50% effective concentration values in the device were much higher than 50% lethal concentration values reported in animal experiments, the tendency of the toxic intensity observed in the former was roughly consistent with that of the acute toxicity in the latter. We further applied the device to evaluate the toxicity of cigarette smoke as an example of actual environmental gases, and successfully measured acute cell death from the gas after 48 h of exposure. The present small device is expected to be one of good tools not only in simultaneously assessing various gaseous chemicals or samples, but also in studying acute toxicity expression mechanisms in human lung epithelia.

Plant response to heavy metal toxicity: comparative study between the hyperaccumulator Thlaspi caerulescens (ecotype Ganges) and nonaccumulator plants: lettuce, radish, and alfalfa.

Thlaspi caerulescens (alpine pennycress) is one of the best-known heavy metal (HM) hyperaccumulating plant species. It exhibits the ability to extract and accumulate various HM at extremely high concentrations. In this hydroponic study, the performance of T. caerulescens (ecotype Ganges) to accumulate Cd, Zn, and Cu was compared with that of three nonaccumulator plants: alfalfa (Medicago sativa), radish (Raphanus sativus), and lettuce (Lactuca sativa). Plants were exposed to the separately dissolved HM salts for 7 days at a wide range of increasing concentrations: 0 (control: 1/5 Hoagland nutrient solution), 0.1, 1, 10, 100, and 1000 microM. The comparative study combined chemical, physiological, and ecotoxicological assessments. Excessive concentrations of HM (100 and 1000 microM) affected plant growth, photosynthesis, and phytoaccumulation efficiency. Root exudation for all plant species was highly and significantly correlated to HM concentration in exposure solutions and proved its importance to counter effect toxicity. T. caerulescens resisted better the phytotoxic effects of Cd and Zn (at 1000 microM each), and translocated them significantly within tissues (366 and 1290 microg g(-1), respectively). At the same HM level, T. caerulescens exhibited lower performances in accumulating Cu when compared with the rest of plant species, mainly alfalfa (298 microg g(-1)). Root elongation inhibition test confirmed the selective aptitude of T. caerulescens to better cope with Cd and Zn toxicities. MetPLATE bioassay showed greater sensitivity to HM toxicity with much lower EC(50) values for beta-galactosidase activity in E. coli. Nevertheless, exaggerated HM concentrations coupled with relatively short exposure time did not allow for an efficient metal phytoextraction thus a significant reduction of ecotoxicity.

Evaluation of mutagenic activities of leachates in landfill sites by micronucleus test and comet assay using goldfish.

To develop a simple system for monitoring the presence of mutagens/carcinogens in the leachates from landfill sites, we used a micronucleus test and a single cell gel electrophoresis (comet) assay originally developed for mice and rats on goldfish (Carassius auratus). The goldfish were exposed for 9 days to the leachate with chemical and biological treatment (treated leachate) or without treatment (raw leachate). The goldfish exposed to several samples died because of the high concentrations of NaCl or ammonium ion (NH4+). In the comet assay using peripheral erythrocytes, the raw leachates showed higher mutagenic activity than the treated leachates. In the micronucleus test, it was difficult to detect the micronuclei in peripheral erythrocytes. On the other hand, the frequency of micronuclei was high in gill cells of goldfish exposed to the raw leachates compared to the treated leachates. A combination of the two bioassays was shown to be useful to evaluate the mutagenic activity of the leachates. We also propose a new scoring method for determination of water quality by using acute toxicity and mutagenic activity.

Development of a genotoxicity detection system using a biosensor.

The umu-lux test is a genotoxicity test using the two genetically modified S. typhmurium TA1535 strains (TL210 and TL210ctl) transformed with the luxCDABE (luciferase gene and fatty acid reductase genes) of Vibrio fischeri as a reporter gene. The TL210 strain detects genotoxicants and the TL210ctl strain detects cytotoxicants. In order to develop a highly sensitive, simple and rapid genotoxicity detection system, we constructed a biosensor using these immobilized strains. The biosensor consists of two immobilized microbial membranes, a sample vessel and photodetectors, and the genotoxicity detection system consists of the biosensor, an isothermal box, a photodetector and an air pump. The total measurement time for genotoxicants using this detection system is about 4 h. When 2% (v/v) DMSO was used as a control, the TL210 strain was not emitting light while the TL210ctl strain was. When 0.3 mg/l 4NQO was used as a genotoxicant, TL210 strain and TL210ctl strain were both emitting light. When HgCl2 was used as a cytotoxicant, neither the TL210 strain nor the TL210ctl strain were emitting light. Therefore, the false negative prevention function of a biosensor using the TL210ctl strain has been checked. These results show that our proposed system can correctly detect genotoxicants.

Response of antioxidative enzymes and apoplastic bypass transport in Thlaspi caerulescens and Raphanus sativus to cadmium stress.

A hydroponics experiment using hyperaccumulator Thlaspi caerulescens (alpine pennycress) and non-specific accumulator Raphanus sativus (common radish) was conducted to investigate the short-term effect of increasing Cd concentrations (0, 25, 50, 75, 100 microM) on metal uptake, chlorophyll content, antioxidative enzymes, and apoplastic bypass flow. As expected, T. caerulescens generally showed better resistance to metal stress, which was reflected by higher Cd accumulation within plant tissues with no signs of chlorosis, or wilt. Glutathione reductase (GR) and superoxide dismutase (SOD) activities in fresh leaves were monitored as the plant metal-detoxifying response. In general, both plant species exhibited an increase trend of GR activity before declining at 100 microM likely due to excessive levels of phytotoxic Cd. SOD activity exhibited almost a similar variation pattern to GR and decreased also at 100 microM Cd. For both plant species, fluorescent PTS uptake (8-hydroxy-1,3,6-pyrenetrisulphonic acid) increased significantly with metal level in exposure solutions indicating that Cd has a comparable effect to drought or salinity in terms of the gain of relative importance in apoplastic bypass transport under such stress conditions.

A rapid and simple evaluation system for gas toxicity using luminous bacteria entrapped by a polyion complex membrane.

We have developed a rapid and simple gas toxicity evaluation system based on bioluminescence inhibition of a marine-derived wild luminous bacterium, Vibrio fischeri. The luminous bacteria were trapped using a thin polyion complex membrane in order to allow semi direct contact between the bacteria and toxic gases. Bioluminescence inhibition ratios of the present system to six reference gases, including benzene, trichloroethylene, acetone, NO(2), SO(2), and CO, were evaluated, and dose-response relationships were successfully obtained after 15 min of gas exposure, except for CO gas. The sensitivity to the five gases except for CO gas of the present system was 1-3 orders of magnitude higher than that in acute animal tests. The present system also allowed for the evaluation of overall toxicity of some environmental gases containing various chemicals. These results clearly demonstrated that the present system would be a valuable prototype for rapid and on-site acute toxicity detection of a gas mixture, such as environmental gases.

Ecotoxicity assessment of sludge and leaching test eluates of sludge.

Sludge mainly collected from wastewater treatment plants were subjected to chemical and ecotoxicological characterization to provide a preliminary assessment of their suitability for land application. We examined the leachability and toxicity of organic compounds in several industrial wastes and recovered materials such as dewatered sludge through standard leaching tests. Several toxicity tests were performed under standard laboratory conditions using a freshwater alga, a crustacean, and some terrestrial plants. Some eluates showed high toxicity. No correlation was found between the ecotoxicological and chemical characteristics or the sum parameters (TOC, total organic carbon concentration). Further, bioassays are extended to exhibit the toxicity, which can be explained by the presence of organic compounds. In general, the eluates differed in their toxicity profiles and their organic chemical composition. Therefore, chemical analyses are unreliable for quantifying biological effects. The samples can be classified as ecotoxic/hazardous based on the LC/EC(50) (lethal concentration/effective concentration reducing 50% of survival or biological activity) values. It is necessary to know the relative ecotoxicity of the potential leachate from organic sludge waste before landfill in order to predict the environmental impact in the event of leakage from the landfill.

Evaluation of the ecotoxicity of solid wastes using rapid leaching test and bioassays.

A rapid leaching test (RLT) involving the use of ultrasonic irradiation was developed. The applicability and suitability of different leaching procedures for solid wastes were evaluated in terms of the leached concentration of heavy metals and total organic carbon and the ecotoxicity potential of the solid wastes. Solid wastes, including incineration ash and sludge, were used. We performed the RLT and the Japanese standard leaching test (JLT-13) to evaluate the ecotoxicity of the solid wastes. The leaching test samples were analyzed using 3 different bioassays. A higher concentration of heavy metals was obtained by the RLT than by JLT-13. The concentrations of organic compounds leached by the RLT correlated well with those of the organic compounds leached by JLT-13. These results indicate that the RLT developed in this study is a useful method to estimate the leachability of heavy metals and organic compounds in solid waste samples. Although the 50% effective concentration (EC(50)) values of the RLT and JLT-13 solid waste eluates derived by a human cell-based bioassay were slightly different, the EC(50) values of these eluates derived by 2 other ecotoxicity tests correlated well.

Determination of volatile fatty acids in landfill leachates by ion-exclusion chromatography.

An ion-exclusion chromatographic method with on-line desalinization for the determination of volatile fatty acids in landfill leachates is described. Highly sensitive conductivity detection of the organic acids was achieved by using dilute p-hydroxybenzoic acid solution as an eluent. Interference with mineral acids was reduced by treatment with barium chloride solution prior to desalinization. A silver-loaded cation-exchange guard column for the desalinization was installed in series with the analytical column to avoid the contamination of organic acids. This method features detection limits of 0.01 mg L(-1) formic acid, 0.02 mg L(-1) acetic acid, 0.05 mg L(-1) propionic acid, and 0.1 mg L(-1) butyric acid, respectively, with an injection of 20 microL sample. Application of the on-line desalinization LC method is illustrated for leachate samples from a Japanese sanitary landfill.