Ethylenediurea (EDU) effects on Japanese larch: an one growing season experiment with simulated regenerating communities and a four growing season application to individual saplings.

Japanese larch (Larix kaempferi (Lamb.) Carr.) and its hybrid are economically important coniferous trees widely grown in the Northern Hemisphere. Ground-level ozone (O3) concentrations have increased since the pre-industrial era, and research projects showed that Japanese larch is susceptible to elevated O3 exposures. Therefore, methodologies are needed to (1) protect Japanese larch against O3 damage and (2) conduct biomonitoring of O3 in Japanese larch forests and, thus, monitor O3 risks to Japanese larch. For the first time, this study evaluates whether the synthetic chemical ethylenediurea (EDU) can protect Japanese larch against O3 damage, in two independent experiments. In the first experiment, seedling communities, simulating natural regeneration, were treated with EDU (0, 100, 200, and 400 mg L-1) and exposed to either ambient or elevated O3 in a growing season. In the second experiment, individually-grown saplings were treated with EDU (0, 200 and 400 mg L-1) and exposed to ambient O3 in two growing seasons and to elevated O3 in the succeeding two growing seasons. The two experiments revealed that EDU concentrations of 200-400 mg L-1 could protect Japanese larch seedling communities and individual saplings against O3-induced inhibition of growth and productivity. However, EDU concentrations ≤ 200 mg L-1 did offer only partial protection when seedling communities were coping with higher level of O3-induced stress, and only 400 mg EDU L-1 fully protected communities under higher stress. Therefore, we conclude that among the concentrations tested the concentration offering maximum protection to Japanese larch plants under high competition and O3-induced stress is that of 400 mg EDU L-1. The results of this study can provide a valuable resource of information for applied forestry in an O3-polluted world.

Effects of Elevated CO2 on Leaf Senescence, Leaf Nitrogen Resorption, and Late-Season Photosynthesis in Tilia americana L.

We investigated the effects of elevated CO2 concentrations ([CO2]) on autumnal leaf falling, late-season photosynthesis, and leaf N resorption efficiency by exposing Tilia americana L. to three CO2 levels (800 ppm A8, 600 ppm A6, and ambient air A4) in nine continuous stirred tank reactors (CSTRs). All leaves were subdivided into the first (Z1), second (Z2), and third bud break (Z3) leaves. Whole plant photosynthesis (PNsat) was determined by summing the products of PNsat and total leaf area in Z1, Z2, and Z3, respectively. The results indicated that 1) the timing of leaf senescence in A8 treatments was 21 days in advance, while the senescence duration sustained 20 days longer than ambient treatment; 2) elevated [CO2] significantly induced the early formation of overwintering buds, with the number increased by 42 and 29% in A8 and A6 treatments, respectively; 3) Z3 leaf photosynthesis increases consistently until the end of the growing season, but Z2 leaves and whole plant showed acclimation when senescence happened; and 4) autumnal N resorption efficiency in A8 and A6 leaves were 25.5% and 22.7% higher than A4, respectively. In conclusion, autumnal senescence of T. americana was accelerated, while the leaf falling duration was extended by elevated [CO2]. The change in leaf phenology makes higher N resorption efficiency and earlier and more winter bud formation possible. Meanwhile, a different response of PNsat within different bud break leaves leads to the disparity between instantaneous measurements of leaf photosynthesis and whole plant photosynthesis in end season.

Commentary: EPA's proposed expansion of dose-response analysis is a positive step towards improving its ecological risk assessment.

The United States Environmental Protection Agency (US EPA) has recently proposed changes to strengthen the transparency of its pivotal regulatory science policy and procedures. In this context, the US EPA aims to enhance the transparency of dose-response data and models, proposing to consider for the first time non-linear biphasic dose-response models. While the proposed changes have the potential to lead to markedly improved ecological risk assessment compared to past and current approaches, we believe there remain open issues for improving the quality of ecological risk assessment, such as the consideration of adaptive, dynamic and interactive effects. Improved risk assessment including adaptive and dynamic non-linear models (beyond classic threshold models) can enhance the quality of regulatory decisions and the protection of ecological health. We suggest that other countries consider adopting a similar scientific-regulatory posture with respect to dose-response modeling via the inclusion of non-linear biphasic models, that incorporate the dynamic potential of biological systems to adapt (i.e., enhancing positive biological endpoints) or maladapt to low levels of stressor agents.

Ethylenediurea (EDU) mitigates the negative effects of ozone in rice: Insights into its mode of action.

Monitoring of ozone damage to crops plays an increasingly important role for the food security of many developing countries. Ethylenediurea (EDU) could be a tool to assess ozone damage to vegetation on field scale, but its physiological mode of action remains unclear. This study investigated mechanisms underlying the ozone-protection effect of EDU in controlled chamber experiments. Ozone sensitive and tolerant rice genotypes were exposed to ozone (108 ppb, 7 hr day-1 ) and control conditions. EDU alleviated ozone effects on plant morphology, foliar symptoms, lipid peroxidation, and photosynthetic parameters in sensitive genotypes. Transcriptome profiling by RNA sequencing revealed that thousands of genes responded to ozone in a sensitive variety, but almost none responded to EDU. Significant interactions between ozone and EDU application occurred mostly in ozone responsive genes, in which up-regulation was mitigated by EDU application. Further experiments documented ozone degrading properties of EDU, as well as EDU deposits on leaf surfaces possibly related to surface protection. EDU application did not mitigate the reaction of plants to other abiotic stresses, including iron toxicity, zinc deficiency, and salinity. This study provided evidence that EDU is a surface protectant that specifically mitigates ozone stress without interfering directly with the plants' stress response systems.

Effects of ozone (O3) and ethylenediurea (EDU) on the ecological stoichiometry of a willow grown in a free-air exposure system.

Ground-level ozone (O3) concentrations have been elevating in the last century. While there has been a notable progress in understanding O3 effects on vegetation, O3 effects on ecological stoichiometry remain unclear, especially early in the oxidative stress. Ethyelenediurea (EDU) is a chemical compound widely applied in research projects as protectant of plants against O3 injury, however its mode of action remains unclear. To investigate O3 and EDU effects early in the stress, we sprayed willow (Salix sachalinensis) plants with 0, 200 or 400 mg EDU L-1, and exposed them to either low ambient O3 (AOZ) or elevated O3 (EOZ) levels during the daytime, for about one month, in a free air O3 controlled exposure (FACE); EDU treatment was repeated every nine days. We collected samples for analyses from basal, top, and shed leaves, before leaves develop visible O3 symptoms. We found that O3 altered the ecological stoichiometry, including impacts in nutrient resorption efficiency, early in the stress. The relation between P content and Fe content seemed to have a critical role in maintaining homeostasis in an effort to prevent O3-induced damage. Photosynthetic pigments and P content appeared to play an important role in EDU mode of action. This study provides novel insights on the stress biology which are of ecological and toxicological importance.

High doses of ethylenediurea (EDU) as soil drenches did not increase leaf N content or cause phytotoxicity in willow grown in fertile soil.

Ground-level ozone (O3) levels are nowadays elevated in wide regions of the Earth, causing significant effects on plants that finally lead to suppressed productivity and yield losses. Ethylenediurea (EDU) is a chemical compound which is widely used in research projects as phytoprotectant against O3 injury. The EDU mode of action remains still unclear, while there are indications that EDU may contribute to plants with nitrogen (N) when the soil is poor in N and the plants have relatively small leaf area. To reveal whether the N content of EDU acts as a fertilizer to plants when the soil is not poor in N and the plants have relatively large total plant leaf area, willow plants (Salix sachalinensis Fr. Schm) were exposed to low ambient O3 levels and treated ten times (9-day interval) with 200mL soil drench containing 0, 800 or 1600mg EDU L-1. Fertilizer was added to a nutrient-poor soil, and the plants had an average plant leaf area of 9.1m2 at the beginning of EDU treatments. Indications for EDU-induced hormesis in maximum electron transport rate (Jmax) and ratio of intercellular to ambient CO2 concentration (Ci:Ca) were observed at the end of the experiment. No other EDU-induced effects on leaf greenness and N content, maximum quantum yield of photosystem II (Fv/Fm), gas exchange, growth and matter production suggest that EDU did not act as N fertilizer and did not cause toxicity under these experimental conditions.

Diagnosing ozone stress and differential tolerance in rice (Oryza sativa L.) with ethylenediurea (EDU).

Rising tropospheric ozone concentrations in Asia necessitate the breeding of adapted rice varieties to ensure food security. However, breeding requires field-based evaluation of ample plant material, which can be technically challenging or very costly when using ozone fumigation facilities. The chemical ethylenediurea (EDU) has been proposed for estimating the effects of ozone in large-scale field applications, but controlled experiments investigating constitutive effects on rice or its suitability to detect genotypic differences in ozone tolerance are missing. This study comprised a controlled open top chamber experiment with four treatments (i) control (average ozone concentration 16 ppb), (ii) control with EDU application, (iii) ozone stress (average 77 ppb for 7 h daily throughout the season), and (iv) ozone stress with EDU application. Three contrasting rice genotypes were tested, i.e. the tolerant line L81 and the sensitive Nipponbare and BR28. The ozone treatment had significant negative effects on plant growth (height and tillering), stomatal conductance, SPAD value, spectral reflectance indices such as the normalized difference vegetation index (NDVI), lipid peroxidation, as well as biomass and grain yields. These negative effects were more pronounced in the a priori sensitive varieties, especially the widely grown Bangladeshi variety BR28, which showed grain yield reductions by 37 percent. EDU application had almost no effects on plants in the absence of ozone, but partly mitigated ozone effects on foliar symptoms, lipid peroxidation, SPAD value, stomatal conductance, several spectral reflectance parameters, panicle number, grain yield, and spikelet sterility. EDU responses were more pronounced in sensitive genotypes than in the tolerant L81. In conclusion, EDU had no constitutive effects on rice and partly offset negative ozone effects, especially in sensitive varieties. It can thus be used to diagnose ozone damage in field grown rice and for distinguishing tolerant (less EDU-responsive) and sensitive (more EDU-responsive) genotypes.

Impacts of ethylenediurea (EDU) soil drench and foliar spray in Salix sachalinensis protection against O3-induced injury.

It is widely accepted that elevated levels of surface ozone (O3) negatively affect plants. Ethylenediurea (EDU) is a synthetic substance which effectively protects plants against O3-caused phytotoxicity. Among other questions, the one still open is: which EDU application method is more appropriate for treating fast-growing tree species. The main aims of this study were: (i) to test if chronic exposure of Salix sachalinensis plants to 200-400mgEDUL-1, the usually applied range for protection against O3 phytotoxicity, is beneficial to plants; (ii) to evaluate the effects of chronic exposure to elevated O3 on S. sachalinensis; (iii) to assess the efficacy of two methods (i.e. soil drench and foliar spray) of EDU application to plants; (iv) to investigate the appropriate concentration of EDU to protect against elevated O3-induced damage in S. sachalinensis; and (v) to compare the two methods of EDU application in terms of effectiveness and EDU consumption. Current-year cuttings grown in infertile soil free from organic matter were exposed either to low ambient O3 (AOZ, 10-h≈28.3nmolmol-1) or to elevated O3 (EOZ, 10-h≈65.8nmolmol-1) levels during daylight hours. Over the growing season, plants were treated every nine days with 200mL soil drench of 0, 200 or 400mgEDUL-1 or with foliar spray of 0, 200 or 400mgEDUL-1 (in two separate experiments). We found that EDU per se had no effects on plants exposed to AOZ. EOZ practically significantly injured S. sachalinensis plants, and the impact was indifferent between the experiments. EDU did not protect plants against EOZ impact when applied as soil drench but it did protect them when applied as 200-400mgL-1 foliar spray. We conclude that EDU may be more effective against O3 phytotoxicity to fast-growing species when applied as a spray than when applied as a drench. Keymessage: Soil-drenched EDU was ineffective in protecting willow plants against O3-induced injury, whereas foliar-sprayed EDU was effective even at the concentration of 200mgL-1.

Moderate drought did not affect the effectiveness of ethylenediurea (EDU) in protecting Populus cathayana from ambient ozone.

A field study was conducted to evaluate the effects of ambient ozone (O3) on an O3-sensitive poplar (Populus cathayana) by using ethylenediurea (EDU) as a chemical protectant under two soil water treatments (well-watered (WW) and moderate drought (MD, 50-60% of WW in volumetric soil water content). EDU was applied as foliar spray at 0, 300, 450, and 600ppm. Photosynthetic parameters, pigment contents, leaf nitrogen, antioxidant capacity, growth, and biomass were measured. The 8h (9:00-17:00) average ambient O3 concentration was 71.7ppb, and AOT40 was 29.2ppmh during the experimental period (9 June to 21 September), which was high enough to cause plant injury. MD had significantly negative effects on P. cathayana, as indicated by reduced photosynthesis, growth, and biomass, and higher MDA contents. On the other hand, EDU significantly increased photosynthesis rate, chlorophyll a fluorescence, Vcmax and Jmax, photosynthetic pigments, total antioxidant capacity, tree growth and biomass accumulation, and reduced lipid peroxidation, but there was no significant interaction between EDU and drought for most parameters, indicating that EDU can efficiently protect Populus cathayana against ambient O3 and the protection was not affected by soil water contents when soil water reached moderate drought level. Among all doses, EDU at 450ppm provided maximum protection. Comparison of EDU-treated and non-treated P. cathayana could be used as a biomarker system in risk assessment of the effects of ambient O3 on forest health.

High doses of ethylene diurea (EDU) are not toxic to willow and act as nitrogen fertilizer.

UNLABELLED: Ethylene diurea (EDU) is synthetic chemical which protects plants against damage caused by ground level O3 and is used experimentally as a biomonitoring tool at doses usually ranging from 200 to 400mgL(-1) a.i. Although several studies have investigated the protective action of EDU, this mechanism remains unclear. Important uncertainties in EDU action are whether EDU acts as a source of nitrogen (N) to plants and whether high doses are phytotoxic. In order to answer these questions, we conducted an open-field experiment where potted willow (Salix sachalinensis Fr. Schm) plants were exposed to ambient O3 conditions and treated with 0, 800 or 1600mgL(-1) EDU as a soil drench, every nine days, for about 2.5months. We examined approximately 50 response variables. Based on N content in different plant organs, we found that (a) all EDU was transferred to the leaves and (b) high doses of EDU increased the leaf N content. However, EDU did not affect the C content and distribution within the plant body. Still, even at the highest dose, EDU was not toxic to this fast-growing species (however such a high dose should not be applied in uncontrolled environments); and there was no EDU persistence in the soil, as indicated by soil N content. Notably, our soil was free from organic matter and N-poor. KEY MESSAGE: EDU per se does not cause toxicity to willow plants when applied as drench to a soil with no organic matter, rather, high EDU doses may act as nitrogen fertilizer in a nitrogen-poor soil.

The first toxicological study of the antiozonant and research tool ethylene diurea (EDU) using a Lemna minor L. bioassay: Hints to its mode of action.

The antiozonant and research tool ethylene diurea (EDU) is widely studied as a phytoprotectant against the widespread pollutant ground-surface ozone. Although it has been extensively used, its potential toxicity in the absence of ozone is unknown and its mode of action is unclear. The purpose of this research was to toxicologically assess EDU and to further investigate its mode of action using Lemna minor L. as a model organism. Application of EDU concentrations greater than 593 mg L(-1) (practically 600 mg L(-1)) resulted in adverse inhibition of colony growth. As no-observed-toxic-effects concentration (NOEL) we recommend a concentration of 296 mg L(-1) (practically 300 mg L(-1)). A hormetic response was detected, i.e. stimulatory effects of low EDU concentrations, which may indicate overcompensation in response to disruption in homeostasis. Growth inhibition and suppressed biomass were associated with impacted chlorophyll a fluorescence (ΦPSII, qP and ETR). Furthermore, EDU increased mesophyll thickness, as indicated by frond succulence index. Applications of concentrations ≥593 mg L(-1) to uncontrolled environments should be avoided due to potential toxicity to sensitive organisms and the environment.

Assessing the effects of ambient ozone in China on snap bean genotypes by using ethylenediurea (EDU).

Four genotypes of snap bean (Phaseolus vulgaris L.) were selected to study the effects of ambient ozone concentration at a cropland area around Beijing by using 450 ppm of ethylenediurea (EDU) as a chemical protectant. During the growing season, the 8h (9:00-17:00) average ozone concentration was very high, approximately 71.3 ppb, and AOT40 was 29.0 ppm.h. All genotypes showed foliar injury, but ozone-sensitive genotypes exhibited much more injury than ozone-tolerant ones. Compared with control, EDU significantly alleviated foliar injury, increased photosynthesis rate and chlorophyll a fluorescence, Vcmax and Jmax, and seed and pod weights in ozone-sensitive genotypes but not in ozone-tolerant genotypes. EDU did not significantly affect antioxidant contents in any of the genotypes. Therefore, EDU effectively protected sensitive genotypes from ambient ozone damage, while protection on ozone-tolerant genotypes was limited. EDU can be regarded as a useful tool in risk assessment of ambient ozone on food security.

Chronic drought stress reduced but not protected Shantung maple (Acer truncatum Bunge) from adverse effects of ozone (O3) on growth and physiology in the suburb of Beijing, China.

A two-year experiment exposing Acer truncatum Bunge seedlings to elevated ozone (O3) concentrations above ambient air (AO) and drought stress (DS) was carried out using open-top chambers (OTCs) in a suburb of Beijing in north China in 2012-2013. The results suggested that AO and DS had both significantly reduced leaf mass area (LMA), stomatal conductance (Gs), light saturated photosynthetic rate (Asat) as well as above and below ground biomass at the end of the experiment. It appeared that while drought stress mitigated the expression of foliar injury, LMA, leaf photosynthetic pigments, height growth and basal diameter, due to limited carbon fixation, the O3 - induced reductions in Asat, Gs and total biomass were enhanced 23.7%. 15.5% and 8.1% respectively. These data suggest that when the whole plant was considered that drought under the conditions of this experiment did not protect the Shantung maple seedlings from the effects of O3.

Projected carbon dioxide to increase grass pollen and allergen exposure despite higher ozone levels.

One expected effect of climate change on human health is increasing allergic and asthmatic symptoms through changes in pollen biology. Allergic diseases have a large impact on human health globally, with 10-30% of the population affected by allergic rhinitis and more than 300 million affected by asthma. Pollen from grass species, which are highly allergenic and occur worldwide, elicits allergic responses in 20% of the general population and 40% of atopic individuals. Here we examine the effects of elevated levels of two greenhouse gases, carbon dioxide (CO2), a growth and reproductive stimulator of plants, and ozone (O3), a repressor, on pollen and allergen production in Timothy grass (Phleum pratense L.). We conducted a fully factorial experiment in which plants were grown at ambient and/or elevated levels of O3 and CO2, to simulate present and projected levels of both gases and their potential interactive effects. We captured and counted pollen from flowers in each treatment and assayed for concentrations of the allergen protein, Phl p 5. We found that elevated levels of CO2 increased the amount of grass pollen produced by ∼50% per flower, regardless of O3 levels. Elevated O3 significantly reduced the Phl p 5 content of the pollen but the net effect of rising pollen numbers with elevated CO2 indicate increased allergen exposure under elevated levels of both greenhouse gases. Using quantitative estimates of increased pollen production and number of flowering plants per treatment, we estimated that airborne grass pollen concentrations will increase in the future up to ∼200%. Due to the widespread existence of grasses and the particular importance of P. pratense in eliciting allergic responses, our findings provide evidence for significant impacts on human health worldwide as a result of future climate change.

Ozone levels in European and USA cities are increasing more than at rural sites, while peak values are decreasing.

Ground-level ozone (O3) levels are usually lower in urban centers than nearby rural sites. To compare trends in O3 levels during the period 1990-2010, we obtained monitoring data from paired urban and rural sites from the European Environment Agency and the US Environmental Protection Agency. Ozone peaks decreased at both station types, with no significant differences between urban and rural stations. Ozone annual averages increased at both urban and rural sites, with a faster rate of increase for urban centers. The overall trend was for convergence between urban and rural O3 data. Ozone levels exceeded the criteria established for the protection of human and vegetation health at both urban and rural sites.

Effects of a three-year exposure to ambient ozone on biomass allocation in poplar using ethylenediurea.

We examined the effect of ambient ozone on visible foliar injury, growth and biomass in field-grown poplar cuttings of an Oxford clone sensitive to ozone (Populus maximoviczii Henry × berolinensis Dippel) irrigated with ethylenediurea (EDU) or water for three years. EDU is used as an ozone protectant for plants. Protective effects of EDU on ozone visible injury were found. As a result, poplar trees grown under EDU treatment increased leaves, lateral branches and root density in the third year, although no significant enhancement of stem height and diameter was found. Ambient ozone (AOT40, 24.6 ppm h; diurnal hourly average, 40.3 ppb) may finally reduce carbon gain by reducing the number of branches, and thus sites for leaf formation, in ozone-sensitive poplar trees under not-limiting conditions.

Ethylenediurea (EDU): a research tool for assessment and verification of the effects of ground level ozone on plants under natural conditions.

Ethylenediurea (EDU) has been widely used to prevent ozone (O(3)) injury and crop losses in crop plants and growth reductions in forest trees. Successful use requires establishing a dose/response curve for EDU and the proposed plant in the absence of O(3) and in the presence of O(3) before initiating multiple applications to prevent O(3) injury. EDU can be used to verify foliar O(3) symptoms in the field, and to screen plants for sensitivity to O(3) under ambient conditions. Despite considerable research, the mode of action of EDU remains elusive. Additional research on the mode of action of EDU in suppressing O(3) injury in plants may also be helpful in understanding the mode of action of O(3) in causing injury in plants.