Ozone risk assessment at Free-Air Controlled Exposure (FACE) experiments: A critical revisit.

Since risk assessments of tropospheric ozone (O3) are crucial for agricultural and forestry sectors, there is a growing body for realistic assessments by a stomatal flux-based approach in Free-Air Controlled Exposure (FACE) facilities. Ozone risks are normally described as relative risks (RRs), which are calculated by assuming the biomass or yield at zero O3 dose as "reference". However, the estimation of the reference biomass or yield is challenging due to a lack of O3-clean-air treatment at the FACEs and the extrapolation without data in a low O3 range increases the bias for estimating the reference values. Here, we reviewed a current methodology for the risk assessment at FACEs and presented a simple and effective way ("modified Paoletti's approach") of defining RRs just using biomass or yield data with a range of expected impacts under the FACE conditions hypothesizing three possible scenarios based on prediction limits using 95% credible intervals (CI) (1. Best fit using the intercept as reference, 2. Optimistic scenario using a lower CI and 3. Worst scenario using an upper CI). As a result, O3-sensitive species show a relatively narrow effect range (optimistic vs. worst scenario) whereas a wide range of response may be possibly taken in resistant species. Showing a possible effect range allows for a comprehensive understanding of the potential risks and its uncertainties related to a species sensitivity to O3. As a supporting approach, we also recommend to use scientifically relevant tools (i.e., ethylenediurea treatments; mechanistic plant models) for strengthening the obtained results for the RRs against O3. Interestingly, the moderately sensitive or resistant species showed non-linear rather than linear dose-response relationships, suggesting a need for the flexible functional form for the risk assessment to properly describe the complex plant response such as hormesis, which depends on their plasticity to O3 stress.

Chronic ozone exposure preferentially modifies root rather than foliar metabolism of date palm (Phoenix dactylifera) saplings.

In their natural environment, date palms are exposed to chronic atmospheric ozone (O3) concentrations from local and remote sources. In order to elucidate the consequences of this exposure, date palm saplings were treated with ambient, 1.5 and 2.0 times ambient O3 for three months in a free-air controlled exposure facility. Chronic O3 exposure reduced carbohydrate contents in leaves and roots, but this effect was much stronger in roots. Still, sucrose contents of both organs were maintained at elevated O3, though at different steady states. Reduced availability of carbohydrate for the Tricarboxylic acid cycle (TCA cycle) may be responsible for the observed reduced foliar contents of several amino acids, whereas malic acid accumulation in the roots indicates a reduced use of TCA cycle intermediates. Carbohydrate deficiency in roots, but not in leaves caused oxidative stress upon chronic O3 exposure, as indicated by enhanced malonedialdehyde, H2O2 and oxidized glutathione contents despite elevated glutathione reductase activity. Reduced levels of phenolics and flavonoids in the roots resulted from decreased production and, therefore, do not indicate oxidative stress compensation by secondary compounds. These results show that roots of date palms are highly susceptible to chronic O3 exposure as a consequence of carbohydrate deficiency.

Exploring new strategies for ozone-risk assessment: A dynamic-threshold case study.

Tropospheric ozone is a dangerous atmospheric pollutant for forest ecosystems when it penetrates stomata. Thresholds for ozone-risk assessment are based on accumulated stomatal ozone fluxes such as the Phytotoxic Ozone Dose (POD). In order to identify the effect of ozone on a Holm oak forest in central Italy, four flux-based ozone impact response functions were implemented and tested in a multi-layer canopy model AIRTREE and evaluated against Gross Primary Productivity (GPP) obtained from observations of Eddy Covariance fluxes of CO2. To evaluate if a clear phytotoxic threshold exists and if it changes during the year, six different detoxifying thresholds ranging between 0 and 5 nmol O3 m-2 s-1 were tested. The use of species-specific rather than more general response functions based on plant functional types (PFT) increased model accuracy (RMSE reduced by up to 8.5%). In the case of linear response functions, a threshold of 1 nmol m-2 s-2 produced the best results for simulations of the whole year, although the tolerance to ozone changed seasonally, with higher tolerance (5 nmol m-2 s-1 or no ozone impact) for Winter and Spring and lower thresholds in Summer and Fall (0-1 nmol m-2 s-1). A "dynamic threshold" obtained by extracting the best daily threshold values from a range of different simulations helped reduce model overestimation of GPP by 213 g C m-2 y-1 and reduce RMSE up to 7.7%. Finally, a nonlinear ozone correction based on manipulative experiments produced the best results when no detoxifying threshold was applied (0 nmol O3 m-2 s-1), suggesting that nonlinear functions fully account for ozone detoxification. The evidence of seasonal changes in ozone tolerance points to the need for seasonal thresholds to predict ozone damage and highlights the importance of performing more species-specific manipulative experiments to derive response functions for a broad range of plant species.

Toward stomatal-flux based forest protection against ozone: The MOTTLES approach.

European standards for the protection of forests from ozone (O3) are based on atmospheric exposure (AOT40) that is not always representative of O3 effects since it is not a proxy of gas uptake through stomata (stomatal flux). MOTTLES "MOnitoring ozone injury for seTTing new critical LEvelS" is a LIFE project aimed at establishing a permanent network of forest sites based on active O3 monitoring at remote areas at high and medium risk of O3 injury, in order to define new standards based on stomatal flux, i.e. PODY (Phytotoxic Ozone Dose above a threshold Y of uptake). Based on the first year of data collected at MOTTLES sites, we describe the MOTTLES monitoring station, together with protocols and metric calculation methods. AOT40 and PODY, computed with different methods, are then compared and correlated with forest-health indicators (radial growth, crown defoliation, visible foliar O3 injury). For the year 2017, the average AOT40 calculated according to the European Directive was even 5 times (on average 1.7 times) the European legislative standard for the protection of forests. When the metrics were calculated according to the European protocols (EU Directive 2008/50/EC or Modelling and Mapping Manual LTRAP Convention), the values were well correlated to those obtained on the basis of the real duration of the growing season (i.e. MOTTLES method) and were thus representative of the actual exposure/flux. AOT40 showed opposite direction relative to PODY. Visible foliar O3 injury appeared as the best forest-health indicator for O3 under field conditions and was more frequently detected at forest edge than inside the forest. The present work may help the set-up of further long-term forest monitoring sites dedicated to O3 assessment in forests, especially because flux-based assessments are recommended as part of monitoring air pollution impacts on ecosystems in the revised EU National Emissions Ceilings Directive.

Ozone-induced stomatal sluggishness changes stomatal parameters of Jarvis-type model in white birch and deciduous oak.

Stomatal ozone flux is closely related to ozone injury to plants. Jarvis-type multiplicative model has been recommended for estimating stomatal ozone flux in forest trees. Ozone can change stomatal conductance by both stomatal closure and less efficient stomatal control (stomatal sluggishness). However, current Jarvis-type models do not account for these ozone effects on stomatal conductance in forest trees. We examined seasonal course of stomatal conductance in two common deciduous tree species native to northern Japan (white birch: Betula platyphylla var. japonica; deciduous oak: Quercus mongolica var. crispula) grown under free-air ozone exposure. We innovatively considered stomatal sluggishness in the Jarvis-type model using a simple parameter, s, relating to cumulative ozone uptake (defined as POD: phytotoxic ozone dose). We found that ozone decreased stomatal conductance of white birch leaves after full expansion (-28%). However, such a reduction of stomatal conductance by ozone fell in late summer (-10%). At the same time, ozone reduced stomatal sensitivity of white birch to VPD and increased stomatal conductance under low light conditions. In contrast, in deciduous oak, ozone did not clearly change the model parameters. The consideration of both ozone-induced stomatal closure and stomatal sluggishness improved the model performance to estimate stomatal conductance and to explain the dose-response relationship on ozone-induced decline of photosynthesis of white birch. Our results indicate that ozone effects on stomatal conductance (i.e. stomatal closure and stomatal sluggishness) are crucial for modelling studies to determine stomatal response in deciduous trees, especially in species sensitive to ozone.

BVOC responses to realistic nitrogen fertilization and ozone exposure in silver birch.

Emission of BVOC (Biogenic Volatile Organic Compounds) from plant leaves in response to ozone exposure (O3) and nitrogen (N) fertilization is poorly understood. For the first time, BVOC emissions were explored in a forest tree species (silver birch, Betula pendula) exposed for two years to realistic levels of O3 (35, 48 and 69 ppb as daylight average) and N (10, 30 and 70 kg ha(-1) yr(-1), applied weekly to the soil as ammonium nitrate). The main BVOCs emitted were: α-pinene, ß-pinene, limonene, ocimene, (E)-4,8-dimethyl-1,3,7-nonatriene (DMNT) and hexanal. Ozone exposure increased BVOC emission and reduced total leaf area. The effect on emission was stronger when a short-term O3 metric (concentrations at the time of sampling) rather than a long-term one (AOT40) was used. The effect of O3 on total leaf area was not able to compensate for the stimulation of emission, so that responses to O3 at leaf and whole-plant level were similar. Nitrogen fertilization increased total leaf area, decreased α-pinene and ß-pinene emission, and increased ocimene, hexanal and DMNT emission. The increase of leaf area changed the significance of the emission response to N fertilization for most compounds. Nitrogen fertilization mitigated the effects of O3 exposure on total leaf area, while the combined effects of O3 exposure and N fertilization on BVOC emission were additive and not synergistic. In conclusion, O3 exposure and N fertilization have the potential to affect global BVOC via direct effects on plant emission rates and changes in leaf area.

Effects of long-term ambient ozone exposure on biomass and wood traits in poplar treated with ethylenediurea (EDU).

This is the longest continuous experiment where ethylenediurea (EDU) was used to protect plants from ozone (O3). Effects of long-term ambient O3 exposure (23 ppm h AOT40) on biomass of an O3 sensitive poplar clone (Oxford) were examined after six years from in-ground planting. Trees were irrigated with either water or 450 ppm EDU. Above (-51%) and below-ground biomass (-47%) was reduced by O3 although the effect was significant only for stem and coarse roots. Ambient O3 decreased diameter of the lower stem, and increased moisture content along the stem of not-protected plants (+16%). No other change in the physical wood structure was observed. A comparison with a previous assessment in the same experiment suggested that O3 effects on biomass partitioning to above-ground organs depend on the tree ontogenetic stage. The root/shoot ratios did not change, suggesting that previous short-term observations of reduced allocation to tree roots may be overestimated.

RUNX1, but not its familial platelet disorder mutants, synergistically activates PF4 gene expression in combination with ETS family proteins.

BACKGROUND: Familial platelet disorder (FPD) is a rare autosomal dominant disease characterized by thrombocytopenia and abnormal platelet function. Causal mutations have been identified in the gene encoding runt-related transcription factor 1 (RUNX1) of FPD patients. OBJECTIVES: To elucidate the role of RUNX1 in the regulation of expression of platelet factor 4 (PF4) and to propose a plausible mechanism underlying RUNX1-mediated induction of the FPD phenotype. METHODS: We assessed whether RUNX1 and its mutants, in combination with E26 transformation-specific-1 (ETS-1), Core-binding factor subunit beta (CBFß), and Friend leukemia virus integration 1 (FLI-1), cooperatively regulate PF4 expression during megakaryocytic differentiation. In an embryonic stem cell differentiation system, expression levels of endogenous and exogenous RUNX1 and PF4 were determined by real-time RT-PCR. Promoter activation by the transcription factors were evaluated by reporter gene assays with HepG2 cells. DNA binding activity and protein interaction were analyzed by electrophoretic mobility shift assay and immunoprecipitation assay with Cos-7 cells, respectively. Protein localization was analyzed by immunocytochemistry and Western blotting with Cos-7 cells. RESULTS: We demonstrated that RUNX1 activates endogenous PF4 expression in megakaryocytic differentiation. RUNX1, but not its mutants, in combination with ETS-1 and CBFß, or FLI-1, synergistically activated the PF4 promoter. Each RUNX1 mutant harbors various functional abnormalities, including loss of DNA-binding activity, abnormal subcellular localization, and/or alterations of binding affinities for ETS-1, CBFß, and FLI-1. CONCLUSIONS: RUNX1, but not its mutants, strongly and synergistically activates PF4 expression along with ETS family proteins. Furthermore, loss of the RUNX1 transcriptional activation function is induced by various functional abnormalities.

[The role and significance of acid suppressive drugs in the eradication of H. pylori].

Recently a new triple therapy with PPI and two antimicrobials is widely accepted instead of classical triple therapy. PPI has direct and indirect effects on H. pylori. Practically a single use of PPI is almost noneffective for eradication of H. pylori. In the combination therapy PPI is supposed to contribute to successful eradication through the inhibition of acid secretion. Because a raise of pH in stomach protects antimicrobials from the degradation by acidic environment. The decrease in the volume of gastric juice also contributes to concentrate antibiotics. Recently interaction between PPI and clarithromycin on metabolic enzyme in liver was reported that explains the synergistic effects of these drugs. The heterogeneity of genotype of metabolic enzyme was also elucidated which might be responsible for the difference in the effect of PPI between the patients. To accomplish successful eradication, full inhibition of acid secretion has to be done. To consider the interactions between PPI and antimicrobials and the heterogeneity of genotype of metabolic enzyme may improve eradication therapy of H. pylori.

Characterization of trace amounts of odorants (sulfur compounds, oxygenates, hydrocarbons, phenols and lower fatty acids) in air in an ICU room.

Characterization of trace amounts of odorants in air in an ICU room (ca.257m2) was carried out by gas chromatography and atmospheric pressure ionization-mass spectrometry (API-MS). The concentrations and odor recognition threshold values of the detected odorants, acetaldehyde, ethanol, n-butyric acid, iso-valeric acid and n-valeric acid are as follows: 44.7 ppb and 15 ppb; 19710 ppb and 6100 ppb; 0.50 ppb and 0.4 ppb; 0.45 ppb and 0.4 ppb; 0.67 ppb and 0.5 ppb, respectively. The detected concentrations of these odorants were significantly higher than the odor recognition threshold values. The compounds may, therefore, be responsible for perception of such odors as mixed odors, body odor, and faint or recognizable alcoholic odor, disinfectant odor, and sour, pungent, and goat odors.

International comparison of odor threshold values of several odorants in Japan and in The Netherlands.

The purpose of this paper is to compare the published odor threshold values of six odorants. In Japan, all of the odor threshold values used in the Offensive Odor Control Law (enacted in 1972) were determined in an odor-free room (4 m3) by a trained panel (20 men, ages 30-45 years who were perfumers) who sniffed the odors directly and made absolute judgments of odor quality and intensity. In The Netherlands, sensorial odor concentration measurements were made with an olfactometer in a mobile sniffing car with eight panelists, four men and four women, ages 18-40 years. Such presentations are repeated with different dilution ratios. Comparison of the threshold data for the six different compounds given as the barely perceptible concentration level revealed striking similarities for hydrogen sulfide (in Japan 0.0005 ppm/in The Netherlands 0.0003 ppm), phenol (0.012/0.010), styrene (0.033/0.016), toluene (0.92/0.99), and tetrachloroethylene (1.8/1.2) but not for m-xylene (0.012/0.12). Such a similarity was not found with any other literature sources.

Gas chromatographic determination of indoles in human mouth and saliva using a flameless alkali sensitized detector (nitrogen/phosphorous-specific detector, NPD).

A simple and sensitive NPD determination of the indoles in the mouth and saliva of a nonsmoker and a smoker was investigated. The present method using NPD detection was found to be suitable for the analysis of the odors in human mouth based on the indoles.

Simple circular odor chart for characterization of trace amounts of odorants discharged from thirteen odor sources.

A simple circular odor chart is proposed for the explanation of the relationship between sensory responses (to odor quality and intensity) to odors and chemical analysis data of the odorants responsible for each odor discharged from thirteen odor sources. The odorants were classified into eight odorant groups and were analyzed by a systematic gas chromatographic (GC) technique. The characterization of the trace amounts of the odorants was carried out by using the values of a new proposed unit (pOU) based on the ratio of detected concentration to recognition threshold value. The calculated pOU values of the eight groups were plotted in circular charts. It was found that the shape and size of each circular odor chart represent the quality and the intensity of each odor.

Simultaneous gas chromatographic analysis of lower fatty acids, phenols and indoles using a glass capillary column.

The simultaneous gas chromatographic separation of a mixture of 14 lower fatty acis, 11 phenols and 7 indoles has been effected by using a glass capillary column. Complete separation of the mixture has been obtained, except for phenol and o-cresol and o-ethylphenol and 3,5-xylenol whose peaks overlapped, and 2- and 3-methylindoles which were poorly separated. The optimum conditions are as follows: stationary phase, FFAP or PEG-20M; column (20 m X 0.28 mm I.D.) temperature, held for 1 min at 145 degrees (FFAP) or 130 degrees (PEG-20M); column oven, heated at 4 degrees/min from 145 or 130 to 180 degrees (FFAP) or 165 degrees (PEG-20M), maintained at 180 or 165 degrees for 30 min; carrier gas (helium) flow-rate, 0.6 ml/min (FFAP), 1.3 ml/min (PEG-20M); flame ionization detector. The method has been applied to the analysis of lower fatty acids, phenols and indoles in Japanese cigarette smoke, where the peaks were identified by the disappearance method using an alkaline pre-column.

Simple and rapid gas-liquid-solid chromatographic analysis of trace concentrations of acetaldehyde in urban air.

A simple and rapid gas-liquid-solid chromatographic method for the analysis of trace concentrations of acetaldehyde in urban air (in the Nagoya area) was developed, with the use of cold trapping with liquid oxygen. In the analytical main column the conditions were: stationary phase, Triton X-100 (0.4%); support, Carbopack B (60-80 mesh); glass column, 1.5 m X 3 mm I.D.; column temperature, 75 degrees; carrier gas (nitrogen) flow-rate, 50-80 ml/min. In the cold trapping pre-column the conditions were: stationary phase, Tris (2-cyanoethoxy) propane (25%); support, Shimalite (AW, DMCS) (60-80 mesh); glass column, 31 cm X 4 mm I.D.; operating temperature for the trapping, -183 degrees (liquid oxygen temperature); operating temperature for injection of the condensed sample into the gas chromatograph, increased from -183 degrees to +100 degrees for 2 min. The acetaldehyde peak was identified by the disappearance method with a 2,4-dinitrophenylhydrazine-orthophosphoric acid-glass beads column. The ranges and average concentrations of acetaldehyde detected in 13 urban air samples were 1.5-9.6 and 4.7 ppb, respectively.

Gas chromatographic determination of styrene as its dibromide.

The selective and sensitive gas chromatographic determination of trace amounts of styrene was studied. Styrene was converted into styrene dibromide by reaction with bromine, and the latter was detected with an electron-capture detector. The minimum detectable amount of the dibromide was about 0.01 ng and the sensitivity to the dibromide was about 500 times higher than the sensitivity of a flame-ionization detector to the styrene monomer. Dodecene-1 and o- and m-cresol gave the greatest interference, but the cresols could easily be removed by reaction with a small amount of a dilute aqueous alkaline solution. The application to a practical specimen, namely urban air, is described.

Gas chromatographic determination of sulphur compounds in town gas.

The gas chromatographic (GC) determination of the sulphur compounds in town gas (in the Nagoya area) was studied by using a flame-photometric detector (FPD) and the cold-trap method with liquid oxygen. The column packings used were 25% TCEP on Shimalite (AW, DMCS), 25% TCP on Shimalite (AW, DMCS), 10% PPE on Shimalite TPA, Porapak Q and silica gel. The major components identified were carbonyl sulphide, hydrogen sulphide, carbon disulphide, thiophene and tetrahydrothiophene (THT). The identities of thiophene and THT were also confirmed by GC combined with the use of a quadrupole mass spectrometer. The average concentrations and standard deviations of thiophene and THT were 8.8 +/- 1.8and 124 +/- 35 ng per 0.051, respectively. The latter value corresponds to 0.7 ppm, which is relatively high for the concentration of an odorant.