Dye-sensitized photodegradation of the fungicide carbendazim and related benzimidazoles

The present work studies the visible-light-promoted photodegradation of the colorless fungicide carbendazim (methyl 2-benzimidazolecarbamate) and several 2-substituted benzimidazoles (SBZ's), in water or water-methanol solution, in the presence of air and, as a photosensitizer, the synthetic xanthene dye Rose Bengal (RB) or the natural pigment riboflavin (Rf). The results indicate that the degradation of each particular SBZ depends on its chemical structure and on the sensitizer employed. In the presence of RB, the degradation always operates via a singlet molecular oxygen (O(2)((1)Delta(g)))-mediated mechanism, through a highly efficient process, as deduced from the comparison of the rate constants for physical and chemical quenching of O(2)((1)Delta(g)). In the presence of Rf, the visible-light irradiation of any of the studied SBZ's produces a series of competitive processes that depend on the relative concentrations of Rf and SBZ. These processes include the quenching of excited singlet and triplet Rf states by the SBZ and the generation of both O(2)((1)Delta(g)) and superoxide radical anion (O(2)(-)), the latter generated by electron transfer from excited Rf species to the dissolved oxygen. The overall result is the photodegradation of the SBZ and the photoprotection of the sensitizer.     

Enhancement of heavy metal accumulation by tissue specific co-expression of iaaM and ACC deaminase genes in plants

The aerobic riboflavin (Rf)-sensitized photodegradation of the endocrine disruptor 4,4'-isopropylidenebisphenol (bisphenol A, BPA), and of the related compounds 4,4'-isopropylidenebis(2,6-dibromophenol) and 4,4'-isopropylidenebis(2,6-dimethylphenol) has been studied in water and water-methanol mixtures through visible-light continuous photolysis, polarographic detection of oxygen uptake, stationary and time-resolved fluorescence spectroscopy, time-resolved near-IR phosphorescence detection and laser flash photolysis techniques. Bisphenols (BPs) quench excited singlet and triplet states of Rf, with rate constants close to the diffusion limit. BPs and dissolved molecular oxygen, employed in similar concentrations, competitively quench triplet excited Rf. As a consequence, superoxide radical anion and singlet molecular oxygen (O(2)((1)Delta(g))) are produced by electron- and energy-transfer processes, respectively, as demonstrated by auxiliary experiments employing selective quenchers of both oxidative species and the exclusive O(2)((1)Delta(g)) generator Rose Bengal. As a global result, the photodegradation of Rf is retarded, whereas BPs are degraded, mainly by an O(2)((1)Delta(g))-mediated mechanism, which constitutes a relatively efficient process in the case of BPA. Oxidation, dimerization and fragmentation products have been identified in the photooxidation of BPA. Results indicate that BPs in natural waters can undergo spontaneous photodegradation under environmental conditions in the presence of adequate photosensitizers.     

Modelling the environmental degradation of water contaminants. Kinetics and mechanism of the riboflavin-sensitised-photooxidation of phenolic compounds

The aerobic visible-light-photosensitised irradiation of methanolic solutions of either of the phenolic-type contaminants model compounds (ArOH) p-phenylphenol (PP), p-nitrophenol (NP) and phenol (Ph), and for two additional phenolic derivatives, namely p-chlorophenol (ClP) and p-methoxyphenol (MeOP), used in some experiments, was carried out. Employing the natural pigment riboflavin (Rf) as a sensitiser, the degradation of both the ArOH and the very sensitiser was observed. A complex mechanism, common for all the ArOH studied, operates. It involves superoxide radical anion (O₂^√−) and singlet molecular oxygen (O₂(¹Δ_g)) reactions. Maintaining Rf in sensitising concentrations levels (≈0.02 mM), the mechanism is highly dependent on the concentration of the ArOH. Kinetic experiments of oxygen and substrate consumption, static fluorescence, laser flash photolysis and time-resolved phosophorescence detection of O₂(¹Δ_g) demonstrate that at ArOH concentrations in the order of 10 mM, no chemical transformation occurs due to the complete quenching of Rf singlet excited state. When ArOH is present in concentrations in the order of mM or lower, O₂^√− is generated from the corresponding Rf radical anion, which is produced by electron transfer reaction from the ArOH to triplet excited Rf. The determined reaction rate constants for this step show a fairly good correlation with the electron-donor capabilities for Ph, PP, NP, ClP and MeOP. In this context, the main oxidative species is O₂^√−, since O₂(¹Δ_g) is quenched in an exclusive physical fashion by the ArOH. The production of O₂^√− regenerates Rf impeding the total degradation of the sensitiser. This kinetic scheme could partially model the fate of ArOH in aquatic media containing natural photosensitisers, under environmental conditions.     

Luminescence investigations of redox cycling of adriamycin.

The light emission from the adriamycin + Co2+ + H2O2 system has been studied. Chemiluminescence, fluorescence and absorption spectra were measured. The fluorescence spectra were time-dependent exhibiting maxima at 555, 590 and 645 nm. The chemiluminescence spectra consist of four bands with maxima at around 460-500, 550-580, 640 and 700 nm. Free radical reaction inhibitors, (1)O2-quenchers and catalase inhibited the light emission indicating that hydroxyl radical, superoxide anion radical and singlet oxygen are generated during the redox cycling of adriamycin. Chemiluminescence studies revealed that adriamycin undergoes chemiexcitation under our experimental conditions.     

Formation of singlet oxygen during farmorubicin oxidation.

The enhanced generation of singlet oxygen (1O2) during oxidation of farmorubicin in the Co(II) + H2O2 system was studied using chemiluminescent, fluorescent and spectrophotometic techniques. The influence of 1O2-quenchers, catalase, superoxide anion radical (O2*-) and hydroxyl radical (HO*) scavengers on the light emission was studied. The spectrophotometric determination of 1O2 was based on bleaching of p-nitrosodimethylaniline caused by an intermediate product of the reaction of 1O2 with imidazole, and was followed by monitoring the decrease in optical density at 440 nm.     

The effect of thymol and its derivatives on reactions generating reactive oxygen species

The effects of thymol (TOH), thymoquinone (TQ) and dithymoquinone (TQ2) on the reactions generating reactive oxygen species (ROS) such as superoxide anion radical (O2*-), hydroxyl radical (HO*) and singlet oxygen (1O2) were tested using the chemiluminescence (CL) and spectrophotometry methods. All tested compounds acted as scavengers of various ROS. The rate constant of 1O2-dimols quenching by thymol was calculated.     

Acetone-induced photodechlorination of Aroclor 1254 in alkaline 2-propanol: Probing the mechanism by thermolysis in the presence of di-t- butyl peroxide

Aroclor 1254 (1860 mg/l) in alkaline 2-propanol and in the presence of acetone (4% v/v) was photodechlorinated to biphenyl at λ >300 nm with exceptionally high quantum yield (Φ= 18). Under similar conditions photodechlorination of extracts of Aroclor 1254 contaminated soil (730 mg/kg) proceeded with lower quantum yield Φ= 0.4. While oxygen severely quenched photolysis, dechlorination was accomplished under thermal conditions using di-t-butyl peroxide, t-BuOOBu-t, as free radical initiator. A free radical chain reaction is suggested in which acetone triplet, T₁(n,π*), or t-BuO^· radical abstracts H-atom from 2-propanol to give the ketyl radical, (CH₃)₂ OH, which after losing a proton to the alkaline medium gives the ketyl radical anion, (CH₃)₂CHO^·−. The Aroclor in turn reacts with the latter species through an electron-transfer process giving unstable aryl radical anion, , which cleaves releasing the chloride anion, Cl⁻ and the aryl radical, A^·     

Charge density of total organic carbon in Finnish lakes

Ion balance calculations were used to estimate the charge density of total organic carbon (TOC) and for calibrating the model of Oliver et al. (1983) for Finnish lakes. The database consisted of samples collected in the autumn of 1987 from 955 randomly selected lakes covering the whole country. Organic anion concentrations were estimated as the difference between Sigma cations and Sigma inorganic anions. The charge density of TOC was estimated by dividing the organic anion concentration by TOC. The median charge density was 7.5 microeq (mg TOC)(-1). In lakes with pH values lower than 4.5 the average charge density was 5.2 microeq (mg TOC)(-1) and it increased to 10.3 microeq (mg TOC)(-1) in lakes with pH higher than or equal to 7.0. The calibrated model explained 79% of the observed variability in organic anion concentration. The value for the total amount of acidic functional groups per mol organic carbon in the model of Oliver et al. (1983) was slightly higher compared to the Finnish calibration. At pH values higher than 4.4 the Finnish calibration gives lower organic anion concentrations compared to the model of Oliver et al. (1983).     

In situ iron activated persulfate oxidative fluid sparging treatment of TCE contamination--a proof of concept study

In situ chemical oxidation (ISCO) is considered a reliable technology to treat groundwater contaminated with high concentrations of organic contaminants. An ISCO oxidant, persulfate anion (S(2)O(8)(2-)) can be activated by ferrous ion (Fe(2+)) to generate sulfate radicals (E(o)=2.6 V), which are capable of destroying trichloroethylene (TCE). The property of polarity inhibits S(2)O(8)(2-) or sulfate radical (SO(4)(-)) from effectively oxidizing separate phase TCE, a dense non-aqueous phase liquid (DNAPL). Thus the oxidation primarily takes place in the aqueous phase where TCE is dissolved. A bench column study was conducted to demonstrate a conceptual remediation method by flushing either S(2)O(8)(2-) or Fe(2+) through a soil column, where the TCE DNAPL was present, and passing the dissolved mixture through either a Fe(2+) or S(2)O(8)(2-) fluid sparging curtain. Also, the effect of a solubility enhancing chemical, hydroxypropyl-beta-cyclodextrin (HPCD), was tested to evaluate its ability to increase the aqueous TCE concentration. Both flushing arrangements may result in similar TCE degradation efficiencies of 35% to 42% estimated by the ratio of TCE degraded/(TCE degraded+TCE remained in effluent) and degradation byproduct chloride generation rates of 4.9 to 7.6 mg Cl(-) per soil column pore volume. The addition of HPCD did greatly increase the aqueous TCE concentration. However, the TCE degradation efficiency decreased because the TCE degradation was a lower percentage of the relatively greater amount of dissolved TCE by HPCD. This conceptual treatment may serve as a reference for potential on-site application.     

Comparison of sludge characteristics and PCR-DGGE based microbial diversity of nanofiltration and microfiltration membrane bioreactors

The objective of this study was to compare the sludge characteristics and microbial community diversity between the submerged nanofiltration membrane bioreactor (NF MBR) and microfiltration membrane bioreactor (MF MBR) treating the same municipal wastewater. The influence of a higher concentration of organic matter and salt was investigated. The results of water qualities showed that the dissolved organic carbon (DOC), total phosphorus (T-P) and salt concentrations of the supernatant in the NF MBR were three, four and two times as high as those in the MF MBR, respectively. The specific oxygen uptake rate of the NF MBR (2.9+/-0.4 mg O(2)g(-1)MLSSh(-1)) was lower than that of the MF MBR (4.3+/-1.1 mg O(2)g(-1)MLSS h(-1)). Result of extractable extracellular polymeric substances showed that the NF MBR sludge had more protein and less polysaccharide compared to the MF MBR sludge, whereas specific amount of total organic carbon were the same in both MBRs. The median floc diameters of the NF MBR and the MF MBR were 72+/-12 microm and 59+/-12 microm, respectively, which could be attributable to the different polysaccharide concentrations between both MBR mixed liquor. A higher concentration of materials (DOC, T-P and salt) in the bioreactor, determined by the high rejection rate of the NF membrane, did not significantly affect the microbial diversity under similar operation conditions.     

Effects of ozone on managed pasture: II. Yield, species composition, canopy structure, and forage quality

Managed pasture composed of grasses, clover and weeds was exposed in open-top chambers to different levels of ozone (O(3)) during two consecutive seasons to study changes in yield, species composition, canopy structure, and forage quality. The pasture was established in 1990 and exposed in 1991 and 1992. Ozone treatments included charcoal-filtered air (CF), non-filtered air (NF), and two treatments with O(3) added to NF air during periods with global radiation >/= 400 W m(-2) (NF(+), NF(++)). The ratio between the 2-year cumulative, radiation-weighted O(3) concentration in ambient air (= 365 microl litre(-1) h) and in the different treatments was 0.50 (CF), 0.85 (NF), 1.11 (NF(+)), and 1.64 (NF(++)). Plots were harvested four times in 1991, and five times in 1992. The total forage yield for both seasons was modified little by O(3). The yield reduction in NF(++) was only 10% as compared to the CF treatment. Also, only marginal changes were observed in forage quality (Ca, crude protein, crude fibre), and in leaf area index and fractional light penetration. Ozone strongly reduced the yield of clover (Trifolium repens L. and Trifolium pratense L.). The O(3)-effect on clover growth was small after the first harvest and increased with each growth period. In NF, the 2-year cumulative clover yield was reduced by 24% relative to CF. In NF(++), clover growth almost ceased near the end of the second season. The reduction in clover yield with increasing O(3) was associated with a slight increase in the yield of grasses (mainly Dacytlis glomerata L). The increase in the proportion of invading species (weeds or herbs) (Taraxacum officinale L.) during the experiment was not significantly affected by O(3). A second order polynomial function was fitted to the data to establish an exposure-response model for the cumulative clover yield and the cumulative, radiation-weighted O(3) dose, and linear models were developed for total forage mass, grass yield and yield of weeds. Reducing O(3) from elevated levels (NF(+) and NF(++)) during the first season to near-ambient levels (NF) during the second season resulted in a significant recovery of clover yield after two re-growth periods. It is concluded that continuous exposure to ambient levels of O(3) negatively affects the yield of clover in frequently cut, managed pasture, but because of the relatively small proportion of clover, the shift in species composition only marginally affects total forage yield and forage quality. It is emphasised, however, that limitations of the experimental system must be taken into account before extrapolations to real field situations can be made.     

Ozone-induced nitrate formation in needles and leaves of Picea abies, Fagus sylvatica and Quercus robur

Much attention has been paid to ozone as a major cause of novel forest decline in Europe. In combination with acidic mist, O(3) has been observed to increase ion leaching. Besides cations lake Mg(2+), Ca(2+), K(+), NH(4)(+), considerable amounts of nitrate were found to be leached by acidic mist from needles of Norway spruce. Controlled fumigation experiments, with 100, 300, and 600 microg O(3)m(-3) over 22 days continuously, have led to a nitrate accumulation of 94.1 +/- 14.8, 119.4 +/- 28.7 and 198.9 +/- 14.9 microg NO(3)(-1) g(-1) FW, respectively, in leaves of Quercus robur. Similar values were found in leaves of Fagus sylvatica and current and previous year needles of Picea abies. Nitrate levels of controls receiving charcoal filtered air were well below 40 microg NO(3)(-) g (-1) FW. Statistically significant elevated nitrate levels were observed after only 48 h of continuous fumigation with 600 microg O(3)m(-3), in all tree species tested, and after 144 h in the 100 microg O(3)m(-3) treatment. In another experiment, trees of Picea abies were kept in two charcoal (C) and two Purafil plus charcoal (P/C) ventilated chambers, and fumigated with O and 500 microg O(3)m(-3) in cabinets of each filter-type in order to eliminate NO(x) from chamber air. After 29 days of continuous ozone fumigation, NO(3)(-) accumulation in needles amounted to 102.0 +/- 37.7 and 137.4 +/- 40.5 microg g(-1) FW in P/C and C-filtered chambers, respectively. Nitrate contents of controls were below 30 microg NO(3)(-)g(-1) FW at the end of the experiment. No significant differences in NO(3)(-) accumulation between filter treatments were observed. Since NO(x) was reduced by more than 95% in the Purafil/charcoal versus the charcoal treatment, NO(3)(-) accumulation in needles can be attributed predominantly to the influence of ozone and not to direct NO(2) uptake of needles by the possible oxidation of NO to NO(2) in the presence of ozone.     

Persulfate oxidation for in situ remediation of TCE. I. Activated by ferrous ion with and without a persulfate-thiosulfate redox couple

The objective of the laboratory study is to examine the conditions under which transition metal ions (e.g., ferrous ion, Fe2+) could activate the persulfate anion (S2O8(2)-) to produce a powerful oxidant known as the sulfate free radical (SO4-*) with a standard redox potential of 2.6 V. The SO4-* is capable of destroying groundwater contaminants in situ such as trichloroethylene (TCE). Experiments using Fe2+ as an activator under various molar ratios of S2O8(2)-/Fe2+/TCE in an aqueous system indicated that partial TCE degradation occurred almost instantaneously and then the reaction stalled. Either destruction of SO4-* in the presence of excess Fe2+ or the rapid conversion of all Fe2+ to Fe3+ limited the ultimate oxidizing capability of the system. Sequential addition of Fe2+ in small increments resulted in an increased TCE removal efficiency. Therefore, it appeared that Fe2+ played an important role in generating SO4-*. An observation of oxidation-reduction potential (ORP) variations revealed that the addition of sodium thiosulfate (Na2S2O3) to the ferrous ion activated persulfate system could significantly decrease the strong oxidizing conditions. It was hypothesized that the thiosulfate induced reducing conditions might convert Fe3+ to a lower valence state of Fe2+, making the Fe2+ available to activate persulfate decomposition. The sequential addition of thiosulfate (S2O3(2)-), after the initial stalling of ferrous ion activated persulfate oxidation of TCE, resulted in an improvement in TCE removal. The ferrous ion activated persulfate-thiosulfate redox couple resulted in fairly complete TCE degradation in aqueous systems in a short time frame. In soil slurry systems, TCE degradation was slower in comparison to aqueous systems.     

Electroassisted methods for waste destruction: silver(II) and peroxydisulfate reagents in the electrochemically mediated oxidation of polyaromatic sulfonates

The electrochemical oxidation is a promising process for organic pollutants which are recalcitrant to biological degradation. The anodic oxidation of 1,5-naphthalenedisulfonic acid, hereafter NDS, was evaluated at a Pt anode using in situ generated Ag(II) cation or peroxydisulfate [S2O(8)(2-)] anion as mediators. Kinetics of the direct chemical oxidation of NDS in the presence of Na2S2O8, and the identification of some oxidation intermediates are also reported. An analysis of the results shows that Ag(II) is characterized by a high-reaction rates, while [S2O(8)(2-)] performs the oxidation of NDS in a complete way.     

Effects of ozone on the grain composition of spring wheat grown in open-top field chambers

In a three-year study carried out at a rural site in Switzerland, spring wheat (Triticum aestivum L. cv. Albis) was exposed to different levels of ozone (O(3)) in open-top-field chambers from the two-leaf stage until harvest. Field plots in ambient air (AA) were used for comparison. Grain recovered from the different treatments was analyzed for minerals (Ca, Mg, K, P), starch, protein, amino acids and alpha-tocopherol, in order to investigate the effect of O(3) on grain composition. Chamber-enclosure had small effects on some parameters (K, protein), but not on others (starch), as shown by the comparison of data from the AA and non-filtered-air treatment (NF). Differences between NF and charcoal-filtered air (CF) were very small. At O(3) concentrations higher than in the NF treatment (O(3)-1 = 1.5xNF and O(3)-2 = 2.5xNF), mineral contents were higher than in the NF and CF treatments. Protein content was increased only in the O(3)-2 treatment. Starch contents decreased from about 63% in the CF treatment to 54% in the O(3)-2 tratment. No effect of O(3) on the content of alpha-tocopherol and on the essential amino acid index of the protein was observed. It is concluded that compositional changes in wheat grain in response to O(3) are minor, and that ambient O(3) is not likely to cause important changes.     

Enhancement by anthraquinone-2-sulphonate of the photonitration of phenol by nitrite: implication for the photoproduction of nitrogen dioxide by coloured dissolved organic matter in surface waters

Anthraquinone-2-sulphonate (AQ2S) under UVA irradiation is able to oxidise nitrite to (·)NO(2) and to induce the nitration of phenol. The process involves the very fast reactions of the excited triplet state (3)AQ2S(*) and its 520-nm absorbing exciplex with water, at different time scales (ns and μs, respectively). Quinones are ubiquitous components of coloured dissolved organic matter (CDOM) in surface waters and AQ2S was adopted here as a proxy of CDOM. Using a recently developed model of surface-water photochemistry, we found that the oxidation of nitrite to (·)NO(2) by (3)CDOM(*) could be an important (·)NO(2) source in water bodies with high [NO(2)(-)] to [NO(3)(-)] ratio, for elevated values of column depth and NPOC.     

2-Chlorophenol induced hydroxyl radical production in mitochondria in Carassius auratus and oxidative stress--an electron paramagnetic resonance study

In our previous study, electron paramagnetic resonance (EPR) evidence of reactive oxygen species (ROS) production in Carassius auratus following 2-chlorophenol (2-CP) administration was provided. To further investigate the potential pathway of ROS production, liver mitochondria of C. auratus was isolated and incubated with 2-CP for 30 min. An EPR analysis indicated ROS was produced, and intensities of ROS increased with increasing concentrations of 2-CP. The ROS was then assigned OH by comparing with Fenton reaction. Either catalase or superoxide dismutase, extinguished OH completely in the mitochondria mixture. These facts suggested that O2(.-) and H2O2 contributed to the formation of OH in mitochondria in C. auratus stressed by 2-CP. Combining previous references and our own data, it is reasonable to suggest that 2-CP is first oxidized by H2O2 present in vivo to form phenoxyl radical under the catalytic action of cellular peroxidase (1); phenoxyl radical oxidizes mitochondria NADH to NAD in the presence of NADH (2); NAD reacts with oxygen in vivo to produce O2(.-) (3); O2(.-) is spontaneously dismutated by SOD to form H2O2 and O2, which creates a renewable supply of H2O2 as the initiators of the chain reactions until NADH is consumed (4); simultaneously with reaction (4), O2(.-) reacts with H2O2 to form OH radical via the Haber-Weiss reaction (5). A strong negative correlation (r=-0.9278, p<0.01) between glutathione (GSH) pool and OH production was observed after fish were i.p. injected with 2-CP (250 mg kg(-1)), indicating the depletion of GSH caused by OH.     

Effects of experimental parameters on NF(3) decomposition fraction in an oxygen-based RF plasma environment

Clean procedure is one of the major emitters of perfluorinated compounds (PFCs) in semiconductor manufacturing. Nitrogen trifluoride (NF(3)) is increasingly the process gas of choice for eliminating PFC emissions. However, its toxic to human and similar global warming potential compared to most other PFCs made NF(3) warranted much more investigation. This study demonstrated a radio-frequency plasma system for decomposing NF(3). The effects of experimental parameters: input power, O(2)/NF(3) ratio, operational pressure and NF(3) feeding concentration on NF(3) decomposition fraction (eta(NF3)) and energy efficiency E(NF3) were examined in detail. The analytical results demonstrated that the NF(3) was almost completely decomposed (>99%) at input power=30W, [NF(3)](in)=1.0% and eta(NF3) increased with input power. However, adding O(2) to the system inhibited NF(3) decomposition and decreased E(NF3). Moreover, eta(NF3) and E(NF3), decreased with gradually increasing operational pressure. Notably, increasing the NF(3) feeding concentration increased molecule density, reducing eta(NF3), but increasing E(NF3). Furthermore, the products detected in the NF(3)/O(2)/Ar plasma system were NO(2), NO, N(2)O, SiF(4), N(2) and F(2). Potential reaction pathways in the oxygen-based NF(3) plasma environment were built-up and elucidated.     

Psidium guajava 'Paluma' (the guava plant) as a new bio-indicator of ozone in the tropics

Psidium guajava 'Paluma' saplings were exposed to carbon filtered air (CF), ambient non-filtered air (NF), and ambient non-filtered air+40ppb ozone (NF+O(3)) 8h per day during two months. The AOT40 values at the end of the experiment were 48, 910 and 12 895ppbh(-1), respectively for the three treatments. After 5 days of exposure (AOT40=1497ppbh(-1)), interveinal red stippling appeared in plants in the NF+O(3) chamber. In the NF chamber, symptoms were observed only after 40 days of exposure (AOT40=880ppbh(-1)). After 60 days, injured leaves per plant corresponded to 86% in NF+O(3) and 25% in the NF treatment, and the average leaf area injured was 45% in NF+O(3) and 5% in the NF treatment. The extent of leaf area injured (leaf injury index) was explained mainly by the accumulated exposure of ozone (r(2)=0.91; p<0.05).     

Tibouchina pulchra (Cham.) Cogn., a native Atlantic Forest species, as a bio-indicator of ozone: visible injury

Tibouchina pulchra saplings were exposed to carbon filtered air (CF), ambient non-filtered air (NF) and ambient non-filtered air+40 ppb ozone (NF+O3) 8 h per day during two months. The AOT40 values at the end of the experiment were 48, 910 and 12,895 ppb h(-1), respectively, for the three treatments. After 25 days of exposure (AOT40=3871 ppb h(-1)), interveinal red stippling appeared in plants in the NF+O3 chamber. In the NF chamber, symptoms were observed only after 60 days of exposure (AOT40=910 ppb h(-1)). After 60 days, injured leaves per plant corresponded to 19% in NF+O3 and 1% in the NF treatment; and the average leaf area injured was 7% within the NF+O3 and 0.2% within the NF treatment. The extent of leaf area injured (leaf injury index) was mostly explained by the accumulated exposure of ozone (r2=0.89; p<0.05).