Effect of ambient-level gas-phase peroxides on foliar injury, growth, and net photosynthesis in Japanese radish (Raphanus sativus)

To investigate the effects of ambient-level gas-phase peroxides concurrent with O₃ on foliar injury, photosynthesis, and biomass in herbaceous plants, we exposed Japanese radish (Raphanus sativus) to clean air, 50 ppb O₃, 100 ppb O₃, and 2-3 ppb peroxides + 50 ppb O₃ in outdoor chambers. Compared with exposure to 100 ppb O₃, exposure to 2-3 ppb peroxides + 50 ppb O₃ induced greater damage in foliar injury, net photosynthetic rates and biomass; the pattern of foliar injury and the cause of net photosynthetic rate reduction also differed from those occurring with O₃ exposure alone. These results indicate for the first time that sub-ppb peroxides + 50 ppb O₃ can cause more severe damage to plants than 100 ppb O₃, and that not only O₃, but also peroxides, could be contributing to the herbaceous plant damage and forest decline observed in Japan's air-polluted urban and remote mountains areas.     

The uptake of O3 by poplar leaves: the impact of a long-term exposure to low O3-concentrations

Poplar shoots were exposed for 3–4 weeks to filtered air, ambient (maximum values 50–60 nl ℓ^-1) or two times ambient O₃-concentrations under controlled environmental conditions in fumigation chambers. A sensitive (Populus nigra ‘Brandaris’) and a tolerant (P. euramericana ‘Robusta’) cultivar were used. At regular intervals the uptake of O₃, transpiration and CO₂ assimilation rate (P_n) of full-grown leaves were measured with leaf cuvettes. For unaffected leaves, the measured flux of O₃ into the leaves appeared to be larger than can be calculated using the stomatal conductance for O₃ (g_s,o) estimated from the transpiration rates of the same leaves. Resistance analysis revealed that the difference was partly a result of a reaction with the external leaf surface. However, when the O₃ flux into the leaf was corrected for this reaction, it was still larger than can be estimated using g_s,o. As a consequence, negative residual or internal resistances (r_i) for O₃ transport into the leaves were assessed. It is postulated that O₃ molecules moving into the leaf follow a shorter pathway than effluxing H₂O-molecules. P. ‘Brandaris’ leaves showed a reduction in P_n after 12 days of exposure to ambient O₃-concentrations, whereas for P. ‘Robusta’ a reduction in P_n was only observed at two times ambient concentrations. A simultaneous decline in the O₃-flux was found in both cases. The decline occurred before a decrease in g_s,o was observed suggesting that the O₃ flux into the affected leaves was first hindered by internal factors. The measured flux of the affected leaves became smaller than the flux estimated using g_s,o and, consequently, positive r_i-values were estimated. The change in r_i suggests that O₃ molecules not only penetrated deeper into the leaf, but also were accumulating at a prolonged exposure. Our results indicate that r_i may be a potentially important component of the overall resistance for O₃-uptake, which may have important consequences for estimating O₃ uptake from water vapour flux data.     

Anthocyanins and tannins in ozone-fumigated guava trees

Psidium guajava “Paluma”, a tropical tree species, is known to be an efficient ozone indicator in tropical countries. When exposed to ozone, this species displays a characteristic leaf injury identified by inter-veinal red stippling on adaxial leaf surfaces. Following 30 days of three ozone treatments consisting of carbon filtered air (CF – AOT40 = 17 ppb h), ambient non-filtered air (NF – AOT40 = 542 ppb h) and ambient non-filtered air + 40 ppb ozone (NF + O₃ – AOT40 = 7802 ppb h), the amounts of residual anthocyanins and tannins present in 10 P. guajava (“Paluma”) saplings were quantified. Higher amounts of anthocyanins were found in the NF + O₃ treatment (1.6%) when compared to the CF (0.97%) and NF (1.30%) (p < 0.05), and of total tannins in the NF + O₃ treatment (0.16%) compared to the CF (0.14%). Condensed tannins showed the same tendency as enhanced amounts. Regression analyses using amounts of tannins and anthocyanins, AOT40 and the leaf injury index (LII), showed a correlation between the leaf injury index and quantities of anthocyanins and total tannins. These results are in accordance with the association between the incidence of red-stippled leaves and ozone polluted environments.     

Relationship between leaf antioxidants and ozone injury in Nicotiana tabacum ‘Bel-W3’ under environmental conditions in São Paulo,SE – Brazil

Previous studies have reported that the extent of leaf injury in Nicotiana tabacum “Bel-W3” exposed to environmental conditions in the city of São Paulo is influenced by weather conditions. This influence may occur by means of antioxidant responses. Thus, this study aimed to evaluate whether daily antioxidant responses to environmental variations interfere on the progression of leaf injury on plants of this cultivar during their exposure in a state park of São Paulo and to determine a linear combination of variables, among antioxidants and environmental factors, which mostly explain this visible response. Plants were exposed at the mentioned site for 14 days in four different experiments. During each experiment, three plants were daily sampled to determine the accumulated percentage of leaf area affected by necrosis and antioxidant responses (concentrations of total ascorbic acid (AA) and activity of superoxide dismutase (SOD) and peroxidases (POD)). Ozone concentrations and weather conditions were also daily measured. Pearson correlations and multivariate analyses assessed the relationship between biological and environmental variables. Leaf injury appeared between the 3rd and 6th days of exposure and increased over the exposure periods. The daily concentrations of AA tended to decrease with time of exposure in all experiments, but the activity of SOD and POD oscillated during plant exposure. Positive correlations were observed between AA or SOD and O₃ concentrations, as well as negative correlations between AA and air temperature. The increasing percentage of leaf necrosis across the whole period was explained by decreasing levels of AA 2 days before injury estimation and by higher O₃ concentrations 5 days before (R² = 0.36; p < 0.001). The use of N. tabacum Bel-W3 as a bioindicator can be restricted by leaf antioxidant responses to both atmospheric contamination and weather conditions.     

Stomatal uptake of O3 in aspen and aspen-birch forests under free-air CO2 and O3 enrichment

Rising atmospheric carbon dioxide (CO₂) may alleviate the toxicological impacts of concurrently rising tropospheric ozone (O₃) during the present century if higher CO₂ is accompanied by lower stomatal conductance (g_s), as assumed by many models. We investigated how elevated concentrations of CO₂ and O₃, alone and in combination, affected the accumulated stomatal flux of O₃ (AFst) by canopies and sun leaves in closed aspen and aspen-birch forests in the free-air CO₂-O₃ enrichment experiment near Rhinelander, Wisconsin. Stomatal conductance for O₃ was derived from sap flux data and AFst was estimated either neglecting or accounting for the potential influence of non-stomatal leaf surface O₃ deposition. Leaf-level AFst (AFst_l) was not reduced by elevated CO₂. Instead, there was a significant CO₂ × O₃ interaction on AFst_l, as a consequence of lower values of g_s in control plots and the combination treatment than in the two single-gas treatments. In addition, aspen leaves had higher AFst_l than birch leaves, and estimates of AFst_l were not very sensitive to non-stomatal leaf surface O₃ deposition. Our results suggest that model projections of large CO₂-induced reductions in g_s alleviating the adverse effect of rising tropospheric O₃ may not be reasonable for northern hardwood forests.     

Spring leaf flush in aspen (Populus tremuloides) clones is altered by long-term growth at elevated carbon dioxide and elevated ozone concentration

Early spring leaf out is important to the success of deciduous trees competing for light and space in dense forest plantation canopies. In this study, we investigated spring leaf flush and how long-term growth at elevated carbon dioxide concentration ([CO₂]) and elevated ozone concentration ([O₃]) altered leaf area index development in a closed Populus tremuloides (aspen) canopy. This work was done at the Aspen FACE experiment where aspen clones have been grown since 1997 in conditions simulating the [CO₂] and [O₃] predicted for ∼2050. The responses of two clones were compared during the first month of spring leaf out when CO₂ fumigation had begun, but O₃ fumigation had not. Trees in elevated [CO₂] plots showed a stimulation of leaf area index (36%), while trees in elevated [O₃] plots had lower leaf area index (−20%). While individual leaf area was not significantly affected by elevated [CO₂], the photosynthetic operating efficiency of aspen leaves was significantly improved (51%). There were no significant differences in the way that the two aspen clones responded to elevated [CO₂]; however, the two clones responded differently to long-term growth at elevated [O₃]. The O₃-sensitive clone, 42E, had reduced individual leaf area when grown at elevated [O₃] (−32%), while the tolerant clone, 216, had larger mature leaf area at elevated [O₃] (46%). These results indicate a clear difference between the two clones in their long-term response to elevated [O₃], which could affect competition between the clones, and result in altered genotypic composition in future atmospheric conditions.     

Diurnal changes in photosynthetic parameters of Populus tremuloides, modulated by elevated concentrations of CO2 and/or O3 and daily climatic variation

The diurnal changes in light-saturated photosynthesis (Pn) under elevated CO₂ and/or O₃ in relation to stomatal conductance (g_s), water potential, intercellular [CO₂], leaf temperature and vapour-pressure difference between leaf and air (VPD_L) were studied at the Aspen FACE site. Two aspen (Populus tremuloides Michx.) clones differing in their sensitivity to ozone were measured. The depression in Pn was found after 10:00 h. The midday decline in Pn corresponded with both decreased g_s and decreased Rubisco carboxylation efficiency, Vc_max. As a result of increasing VPD_L, g_s decreased. Elevated [CO₂] resulted in more pronounced midday decline in Pn compared to ambient concentrations. Moreover, this decline was more pronounced under combined treatment compared to elevated CO₂ treatment.The positive impact of CO₂ on Pn was relatively more pronounced in days with environmental stress but relatively less pronounced during midday depression. The negative impact of ozone tended to decrease in both cases.     

Effects of ozone on growth, yield and leaf gas exchange rates of four Bangladeshi cultivars of rice (Oryza sativa L.)

To assess the effects of tropospheric O₃ on rice cultivated in Bangladesh, four Bangladeshi cultivars (BR11, BR14, BR28 and BR29) of rice (Oryza sativa L.) were exposed daily to charcoal-filtered air or O₃ at 60 and 100 nl l⁻¹ (10:00-17:00) from 1 July to 28 November 2008. The whole-plant dry mass and grain yield per plant of the four cultivars were significantly reduced by the exposure to O₃. The exposure to O₃ significantly reduced net photosynthetic rate of the 12th and flag leaves of the four cultivars. The sensitivity to O₃ of growth, yield and leaf gas exchange rates was not significantly different among the four cultivars. The present study suggests that the sensitivity to O₃ of yield of the four Bangladeshi rice cultivars is greater than that of American rice cultivars and is similar to that of Japanese rice cultivars and that O₃ may detrimentally affect rice production in Bangladesh.     

Biogenic VOC emissions from fresh leaf mulch and wood chips of Grevillea robusta (Australian Silky Oak)

The emissions of VOC from freshly cut and shredded Grevillea robusta (Australian Silky Oak) leaves and wood have been measured. The VOC emissions from fresh leaf mulch and wood chips lasted typically for 30 and 20 h respectively, and consisted primarily of ethanol, (E)-2-hexenal, (Z)-3-hexen-1-ol and acetaldehyde. The integrated emissions of the VOCs were 0.38±0.04 g kg⁻¹ from leaf mulch, and 0.022±0.003 g kg⁻¹ from wood chips. These emissions represent a source of VOCs in urban and rural air that has previously been unquantified and is currently unaccounted for. These VOCs from leaf mulch and wood chips will contribute to both urban photochemistry and secondary organic aerosol formation. Any CH₄ emissions from leaf mulch and wood chips were <1×10⁻¹¹ g g dry mass⁻¹ s⁻¹.     

Measurement and analysis of atmospheric ammonia emissions from anaerobic lagoons

Ammonia-nitrogen flux (NH₃-N=(14/17)NH₃) was determined from six anaerobic swine waste storage and treatment lagoons (primary, secondary, and tertiary) using the dynamic chamber system. Measurements occurred during the fall of 1998 through the early spring of 1999, and each lagoon was examined for approximately one week. Analysis of flux variation was made with respect to lagoon surface water temperature (∼15 cm below the surface), lagoon water pH, total aqueous phase NH_x(=NH₃+NH₄⁺) concentration, and total Kjeldahl nitrogen (TKN). Average lagoon temperatures (across all six lagoons) ranged from approximately 10.3 to 23.3^°C. The pH ranged in value from 6.8 to 8.1. Aqueous NH_x concentration ranged from 37 to 909 mg N l⁻¹, and TKN varied from 87 to 950 mg N l⁻¹. Fluxes were the largest at the primary lagoon in Kenansville, NC (March 1999) with an average value of 120.3 μg N m⁻² min⁻¹, and smallest at the tertiary lagoon in Rocky Mount, NC (November 1998) at 40.7 μg N m⁻² min⁻¹. Emission rates were found to be correlated with both surface lagoon water temperature and aqueous NH_x concentration. The NH₃-N flux may be modeled as ln(NH₃-N flux)=1.0788+0.0406T_L+0.0015([NH_x]) (R²=0.74), where NH₃-N flux is the ammonia flux from the lagoon surface in μg N m⁻² min⁻¹, T_L is the lagoon surface water temperature in ^°C, and [NH_x] is the total ammonia-nitrogen concentration in mg N l⁻¹.     

Leaf size and surface characteristics of Betula papyrifera exposed to elevated CO2 and O3

Betula papyrifera trees were exposed to elevated concentrations of CO₂ (1.4 × ambient), O₃ (1.2 × ambient) or CO₂ + O₃ at the Aspen Free-air CO₂ Enrichment Experiment. The treatment effects on leaf surface characteristics were studied after nine years of tree exposure. CO₂ and O₃ increased epidermal cell size and reduced epidermal cell density but leaf size was not altered. Stomatal density remained unaffected, but stomatal index increased under elevated CO₂. Cuticular ridges and epicuticular wax crystallites were less evident under CO₂ and CO₂ + O₃. The increase in amorphous deposits, particularly under CO₂ + O_3, was associated with the appearance of elongated plate crystallites in stomatal chambers. Increased proportions of alkyl esters resulted from increased esterification of fatty acids and alcohols under elevated CO₂ + O₃. The combination of elevated CO₂ and O₃ resulted in different responses than expected under exposure to CO₂ or O₃ alone.     

Effects of chronic elevated ozone exposure on gas exchange responses of adult beech trees (Fagus sylvatica) as related to the within-canopy light gradient

The effects of elevated O₃ on photosynthetic properties in adult beech trees (Fagus sylvatica) were investigated in relation to leaf mass per area as a measure of the gradually changing, within-canopy light availability. Leaves under elevated O₃ showed decreased stomatal conductance at unchanged carboxylation capacity of Rubisco, which was consistent with enhanced δ¹³C of leaf organic matter, regardless of the light environment during growth. In parallel, increased energy demand for O₃ detoxification and repair was suggested under elevated O₃ owing to enhanced dark respiration. Only in shade-grown leaves, light-limited photosynthesis was reduced under elevated O₃, this effect being accompanied by lowered F_v/F_m. These results suggest that chronic O₃ exposure primarily caused stomatal closure to adult beech trees in the field regardless of the within-canopy light gradient. However, light limitation apparently raised the O₃ sensitivity of photosynthesis and accelerated senescence in shade leaves.     

Stomatal characteristics and infection biology of Pyrenopeziza betulicola in Betula pendula trees grown under elevated CO2 and O3

Two silver birch clones were exposed to ambient and elevated concentrations of CO₂ and O₃, and their combination for 3 years, using open-top chambers. We evaluated the effects of elevated CO₂ and O₃ on stomatal conductance (g_s), density (SD) and index (SI), length of the guard cells, and epidermal cell size and number, with respect to crown position and leaf type. The relationship between the infection biology of the fungus (Pyrenopeziza betulicola) causing leaf spot disease and stomatal characteristics was also studied. Leaf type was an important determinant of O₃ response in silver birch, while crown position and clone played only a minor role. Elevated CO₂ reduced the g_s, but had otherwise no significant effect on the parameters studied. No significant interactions between elevated CO₂ and O₃ were found. The infection biology of P. betulicola was not correlated with SD or g_s, but it did occasionally correlate positively with the length of the guard cells.     

Cutleaf coneflower (Rudbeckia laciniata L.) response to ozone and ethylenediurea (EDU)

Cutleaf coneflower (Rudbeckia laciniata L.) seedlings were placed into open-top chambers in May, 2004 and fumigated for 12 wks. Nine chambers were fumigated with either carbon-filtered air (CF), non-filtered air (NF) or twice-ambient (2×) ozone (O₃). Ethylenediurea (EDU) was applied as a foliar spray weekly at 0 (control), 200, 400 or 600 ppm. Foliar injury occurred at ambient (30%) and elevated O₃ (100%). Elevated O₃ resulted in significant decreases in biomass and nutritive quality. Ethylenediurea reduced percent of leaves injured, but decreased root and total biomass. Foliar concentrations of cell-wall constituents were not affected by EDU alone; however, EDU × O₃ interactions were observed for total cell-wall constituents and lignocellulose fraction. Our results demonstrated that O₃ altered the physiology and productivity of cutleaf coneflower, and although reducing visible injury EDU may be phytotoxic at higher concentrations.     

A flux-based assessment of the effects of ozone on foliar injury, photosynthesis, and yield of bean (Phaseolus vulgaris L. cv. Borlotto Nano Lingua di Fuoco) in open-top chambers

Stomatal ozone uptake, determined with the Jarvis' approach, was related to photosynthetic efficiency assessed by chlorophyll fluorescence and reflectance measurements in open-top chamber experiments on Phaseolus vulgaris. The effects of O₃ exposure were also evaluated in terms of visible and microscopical leaf injury and plant productivity. Results showed that microscopical leaf symptoms, assessed as cell death and H₂O₂ accumulation, preceded by 3-4 days the appearance of visible symptoms. An effective dose of ozone stomatal flux for visible leaf damages was found around 1.33 mmol O₃ m⁻². Significant linear dose-response relationships were obtained between accumulated fluxes and optical indices (PRI, NDI, ΔF/F_m^′). The negative effects on photosynthesis reduced plant productivity, affecting the number of pods and seeds, but not seed weight. These results, besides contributing to the development of a flux-based ozone risk assessment for crops in Europe, highlight the potentiality of reflectance measurements for the early detection of ozone stress.     

Validation of the stomatal flux approach for the assessment of ozone visible injury in young forest trees. Results from the TOP (transboundary ozone pollution) experiment at Curno, Italy

This paper summarises some of the main results of a two-year experiment carried out in an Open-Top Chambers facility in Northern Italy. Seedlings of Populus nigra, Fagus sylvatica, Quercus robur and Fraxinus excelsior have been subjected to different ozone treatments (charcoal-filtered and non-filtered air) and soil moisture regimes (irrigated and non-irrigated plots). Stomatal conductance models were applied and parameterised under South Alpine environmental conditions and stomatal ozone fluxes have been calculated.The flux-based approach provided a better performance than AOT40 in predicting the onset of foliar visible injuries. Critical flux levels, related to visible leaf injury, are proposed for P. nigra and F. sylvatica (ranging between 30 and 33 mmol O₃ m⁻²). Soil water stress delayed visible injury appearance and development by limiting ozone uptake. Data from charcoal-filtered treatments suggest the existence of an hourly flux threshold, below which may occur a complete ozone detoxification.     

Effects of ozone on growth, yield and leaf gas exchange rates of two Bangladeshi cultivars of wheat (Triticum aestivum L.)

To clarify the effects of O₃ on crop plants cultivated in Bangladesh, two Bangladeshi wheat cultivars (Sufi and Bijoy) were grown in plastic boxes filled with Andisol and exposed daily to charcoal-filtered air or O₃ at 60 and 100 nl l⁻¹ (10:00-17:00) from 13 March to 4 June 2008. The whole-plant dry mass and grain yield per plant of the two cultivars at the final harvest were significantly reduced by the exposure to O₃. Although there was no significant effect of O₃ on stomatal diffusive conductance to H₂O of flag leaf, net photosynthetic rate of the leaf was significantly reduced by the exposure to O_3. The sensitivity of growth, yield, yield components and leaf gas exchange rates to O₃ was not significantly different between the two cultivars. The results obtained in the present study suggest that ambient levels of O₃ may detrimentally affect wheat production in Bangladesh.     

The annual variation in stomatal ammonia compensation point of rye grass (Lolium perenne L.) leaves in an intensively managed grassland

The stomatal ammonia compensation point for ammonia (NH₃) of an intensively managed pasture of rye grass (Lolium perenne L.) was followed from mid January till November 2000. Leaf samples were taken every week. Simultaneously, the ambient NH₃ concentration was measured. Meteorological data (temperature, wind speed, rainfall and radiance) were collected from a nearby field station. The vacuum infiltration technique was used to isolate the apoplastic solution of the leaves. From the determined ammonium (NH₄⁺) concentration and pH in the apoplast, the gaseous NH₃ concentration inside the leaves was calculated, i.e. the so-called stomatal compensation point (χ_s).Temperature appeared to have a predominant effect on χ_s, partly by affecting the equilibrium between gaseous NH₃ inside the leaf and NH₃ dissolved in the apoplast and partly by affecting physiological processes influencing the NH₄⁺ concentration in the apoplast. Results of the present study suggest that these temperature effects were counteracting. On one hand temperature increase during early spring stimulated NH₃ volatilisation from the apoplast, on the other hand it led to a decline in apoplastic NH₄⁺ from 0.9 to 0.2 mM, thereby diminishing the emission potential of the leaf. The low NH₄⁺ concentrations during spring and summer coincided with a low total leaf N content (<3% dw). However, there was no clear relationship between these two variables. The total N content of the leaf tissue is therefore an inadequate parameter for prediction of the potential NH₃ emission from rye grass leaves. No annual trend was found for the apoplast pH. With a few exceptions, pH varied between 5.9 and 6.5 throughout the experimental period.The calculated values for χ_s varied between 0.5 and 4 μg m⁻³. The gaseous NH₃ concentrations inside the grass leaves were, with a few exceptions, always smaller than the measured ambient NH₃ concentrations. The present study indicates that under the current ambient NH₃ concentrations in the Netherlands, the grass canopy is unlikely to be a major source of NH₃ emission.     

Elevated ozone phytotoxicity ameliorations in mung bean {Vigna radiata (L.) Wilczek} by foliar nebulization of silicic acid and ascorbic acid

The present work provides an insight into the development of biochemical adaptations in mung beans against ozone (O₃) toxicity. The study aims to explore the O₃ stress tolerance potential of mung bean genotypes under exogenous application of growth regulators. The seeds of twelve mung bean genotypes were grown in plastic pots under controlled conditions in the glasshouse. Six treatments, control (ambient ozone level 40–45 ppb), ambient O₃ with ascorbic acid, ambient ozone with silicic acid, elevated ozone (120 ppb), elevated O₃ with ascorbic acid (10 mM), and elevated ozone with silicic acid (0.1 mM) were applied. The O₃ fumigation was carried out using an O₃ generator. The results revealed that ascorbic acid and silicic acid application decreased the number of plants with foliar O₃ injury symptoms in different degrees, i.e., zero, first, second, third, and fourth degrees; whereas 0–4 degree symptoms represent, no symptoms, symptoms occupying?<?1/4, 1/4–1/2, 1/2–3/4, and?>?3/4 of the total foliage area, respectively. Application of ascorbic acid and silicic acid also prevented the plants from the negative effects of O₃ in terms of fresh as well as dry matter production, leaf chlorophyll, carotenoids, soluble proteins and ascorbic acid, proline, and malondialdehyde (MDA) contents. Overall, silicic acid application proved more effective in reducing the negative effects of O₃ on mung bean genotypes as compared to that of the ascorbic acid. Three mung bean genotypes (NM 20–21, NM-2006, and NM-2016) were identified to have a better adaptive mechanism for O₃ toxicity tolerance and may be good candidates for future variety development programs.

     

Elevated CO2 response of photosynthesis depends on ozone concentration in aspen

The effect of elevated CO₂ and O₃ on apparent quantum yield (?), maximum photosynthesis (P_max), carboxylation efficiency (V_cmax) and electron transport capacity (J_max) at different canopy locations was studied in two aspen (Populus tremuloides) clones of contrasting O₃ tolerance. Local light climate at every leaf was characterized as fraction of above-canopy photosynthetic photon flux density (%PPFD). Elevated CO₂ alone did not affect ? or P_max, and increased J_max in the O₃-sensitive, but not in the O₃-tolerant clone. Elevated O₃ decreased leaf chlorophyll content and all photosynthetic parameters, particularly in the lower canopy, and the negative impact of O₃ increased through time. Significant interaction effect, whereby the negative impact of elevated O₃ was exaggerated by elevated CO₂ was seen in Chl, N and J_max, and occurred in both O₃-tolerant and O₃-sensitive clones. The clonal differences in the level of CO₂ × O₃ interaction suggest a relationship between photosynthetic acclimation and background O₃ concentration.