Gas exchange, growth, and chemical parameters in a native Atlantic forest tree species in polluted areas of Cubatão, Brazil

Gas exchange, growth, and chemical parameters in a native Atlantic forest tree species in polluted areas of Cubatão, Brazil

Ecotoxicology and Environmental Safety 54 (2003) 339–345 Gas exchange, growth, and chemical parameters in a native Atlantic forest tree species in po...

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Ecotoxicology and Environmental Safety 54 (2003) 339–345

Gas exchange, growth, and chemical parameters in a native Atlantic forest tree species in polluted areas of Cubata˜o, Brazil R.M. Moraes,a,* W.B.C. Delitti,b and J.A.P.V. Moraesc a Instituto de Botaˆnica, Caixa Postal 4005, 01061-970 Sa˜o Paulo, SP, Brazil Universidade de Sa˜o Paulo, Caixa Postal 11461, 05422-970 Sa˜o Paulo, SP, Brazil c Universidade Federal de Sa˜o Carlos, Caixa Postal 676, CEP 13565-905, Sa˜o Carlos, SP, Brazil b

Received 27 November 2001; received in revised form 11 September 2002; accepted 20 September 2002

Abstract The Atlantic forest species near the industrial complex of Cubata˜o, Brazil have been subjected to heavy air pollution for decades. In this study, we used some physiological parameters (gas exchange, growth and chemical contents) to biomonitor the effects of air pollution on Tibouchina pulchra, one of the most common tree species in this forest. Under standardized conditions, saplings were exposed to the environment from April to July and from July to September of 1998, at three different sites in the vicinity of the industrial complex: the Valley of Pilo˜es River (VP), the control area; the Valley of Mogi River (VM), near fertilizer, metallurgical, and cement industries sustaining high concentrations of fluorides, N and S oxides, and particulate materials; and Caminho do Mar (CM), near petrochemical industries under N and S oxides, photooxidants, and organic compounds. Plants exposed to CM and VM conditions presented visible injuries, reductions in net photosynthesis, growth parameters, and ascorbate concentrations, and increased F, N, and S foliar concentrations. These results indicate that the environmental conditions around these industries are still harmful to plants. r 2003 Elsevier Science (USA). All rights reserved. Keywords: Tibouchina pulchra; Air pollution; Biomonitoring; Gas exchange; Growth; Fluoride

1. Introduction The city of Cubata˜o, in southeast Brazil, maintains an industrial complex composed of 23 large industries, including a steel mill, an oil refinery, 7 fertilizer plants, a cement plant and 11 chemical/petrochemical plants, adding up to 260 pollutant emission sources. Despite recent reductions, air pollutant concentrations remain high and frequently exceed Brazilian air quality standards. In 1998, industrial emissions in Cubata˜o amounted to 59,000 tons of particulate materials, 11,000 tons of volatile hydrocarbons, 8000 tons of NOx, 35,000 tons of SO2, 28 tons of gaseous fluorides, and 44 tons of ammonia (CETESB, 1999). Secondary pollutants, such as O3 and peroxyacetyl nitrate (PAN), also reach significant levels in some areas of Cubata˜o (Klumpp et al., 1994). The environmental problems caused by the industrial activities are aggravated by the climate and topography *Corresponding author. Fax: +55-11-5073-3678. E-mail address: [email protected] (R.M. Moraes).

of the site, unfavorable to pollutant dispersion. Cubata˜o is located in a narrow coastal plain surrounded by a steep mountain range to the north, west, and east and by the sea to the south. During the day, the winds blow from the sea to the continent, carrying pollutants to the mountains, where they are channeled into narrow valleys. Thermal inversions often occur in winter months (CETESB, 1999). For decades, the Atlantic forest that covers the hillsides surrounding Cubata˜o has been affected by air pollutants emitted from the industrial complex, and the impact on the vegetation continues to the present (Domingos et al., 1998). Various studies to verify the phytotoxicity of the air pollutants in this area were performed, initially employing standardized indicator plants with known sensitivity or accumulative capacity such as Nicotiana tabacum, Petunia hybrids, Urtica urens, Gladiolus hybrids, and Lolium multiflorum ssp. italicum (Klumpp et al., 1994; Domingos et al., 1998). In addition, experiments with native tree species, such as Tibouchina pulchra, Miconia pyrifolia, and Cecropia

0147-6513/03/$ - see front matter r 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0147-6513(02)00067-2


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glazioui (Domingos et al., 1998; Klumpp et al., 2000) aiming to reinforce conclusions on the causes of vegetation damage and to facilitate risk prognosis were conducted. T. pulchra proved to be an adequate accumulative indicator for fluoride, sulfur, and metals, with high accumulation capacity (Klumpp et al., 2000). Nevertheless, although there are numerous studies describing the harmful influence of air pollutants on the CO2 assimilation rate and on other physiological parameters in plants from the temperate regions, these responses are rarely described for tropical species. For example, whether the high absorption of toxic elements by T. pulchra exposed to pollutant emissions from industrial plants in the Cubata˜o region is altering its net photosynthesis rate and to what extent this might be related to growth disturbances are unknown. This study was thus undertaken to evaluate the changes caused by different types and levels of pollutants on saplings of T. pulchra Cogn. This species is found throughout the Atlantic forest region and is of major importance in the definition of vegetation physiognomy and structure (Leita˜o Filho et al., 1993); it is also considered resistant to air pollution (Domingos et al., 1998).

2. Materials and methods The city of Cubata˜o is located at 231450 –231550 S and 461210 –461300 W, southeast Brazil. The climate is tropical and cloudy with no dry season and shows high annual averages for temperature (231C) and precipitation (2600 mm). In the region of Cubata˜o the distribution of the industries, air circulation, and mass transport delimit areas under distinct air pollution influences (Klumpp et al., 1994). Based on these, three sampling sites— Valley of Mogi (VM), Caminho do Mar (CM), and Valley of Pilo˜es (VP) (Fig. 1)—were selected at similar altitudes and meteorological conditions, differing, however, with respect to their air pollution influence (Klumpp et al., 1994; CETESB, 1999). The VM site is located at the entrance of the Mogi River Valley, an area downwind from the industries of Cubata˜o, which presents severely damaged vegetation. VM is situated around 20 m above sea level (asl) and presents mean annual precipitation of 2500 mm, mean annual temperature of 221C, and mean annual relative humidity of 84% (Furlan et al., 1999). It is situated close to the core of the industrial complex and is under the influence of pollutants from the fertilizer, metallurgical,

Fig. 1. Map of the research area with the location of the exposure sites and the emission sources (Klumpp et al., 1994).

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and cement industries. Particulate matter, fluorides, and compounds of sulfur and nitrogen have heavily polluted this area (Klumpp et al., 1994; Jaeschke, 1997). According to CETESB (1999), in 1998 the industries in this region emitted 8,200 tons of SO2, 12,751 tons of NOx, 31,056 tons of particulate materials, 108 tons of fluoride and 113 tons of ammonia. The local Atlantic rain forest has been changed to a secondary community with a decreased number of species, genera, and families of vascular plants (Leita˜o Filho et al., 1993). The CM site is located at an altitude of 80 m asl, with annual averages of temperature, relative humidity, and precipitation of 221C, 84%, and 2800 mm, respectively (Furlan et al., 1999). CM is situated near oil refinery and petrochemical plants. The area is subjected to high levels of ozone, PAN, sulfur dioxide, volatile hydrocarbons, and nitrogen oxides (Klumpp et al., 1994; Jaeschke, 1997). In 1998, the industries in this region emitted 7,033 tons of SO2, 3,767 tons of NOx, 2,391 tons of hydrocarbons and 7,570 tons of particulate materials. The vegetation in this area presents severe damage. As in the case of VM, the Atlantic rain forest in the vicinity of CM presents reduced biodiversity as compared with native unpolluted sites. The VP site was chosen as the control area. It is located in the Pilo˜es River Valley, southwest of the industrial complex, at 40 m asl. VP has an annual precipitation of 3000 mm and an annual mean temperature of 231C (Furlan et al., 1999). Klumpp et al. (1994) characterized the Cubata˜o region according to the distribution of the different types of pollutants, based on analyses of visible leaf damages and leaf chemical contents in standard species (N. tabacum Bel W3, U. urens, Petunia hybrida cv. Mirage, Gladiolus hybrida. cv. White friendship, L. multiflorum italicum cv. Lema). They concluded that the pollution level was very low in VP and came mainly from vehicles. In addition to the absence of visible damages, data on phytotoxical element concentrations, biochemical changes, phenolic compound concentrations, and growth rates reconfirm the low level of air pollution at VP (Klumpp et al., 1996a, b, 1997, 1998, 2000; Furlan et al., 1999). This air quality was not observed in CM and VM, where pollution levels were very high. Geographic barriers and a location out of the way of the land-to-sea breezeway prevent air pollutants from the industrial complex from reaching VP (Jaeschke, 1997). The vegetation presents no signs of degradation induced by air pollution (Leita˜o Filho et al., 1993). Monitoring campaigns have demonstrated that the air pollutant concentrations in the Pilo˜es Valley are below those established in Brazilian air quality legislation, which sets limits of 150 mg/m3 of inhalable particles and total suspended particles (24 h), 100 mg/m3 of SO2 (24 h), 160 mg/m3 of O3 (1 h), and 190 mg/m3 of NO2 (1 h). Because of this, the State Agency of Air Quality Control


(CETESB) does not continuously monitor the air quality in this area. From 1991 to 1994, for example, the daily average fluoride concentrations in VP were under 0.1 mg/m3 while at VM they varied from 0.5 to 2.5 mg/m3. At the same time, the average daily 1-h maximum concentrations of SO2, O3, and NO2 in VP were (in mg/m3) 35, 63, and 18, respectively, while they were 70, 69, and 67 mg/m3 at the VM site and 128, 151, and 49 mg/m3 at the CM site (Klumpp et al., 2000). T. pulchra Cogn. belongs to the Melastomataceae family. It is a tree species native to southeast Brazilian Atlantic rain forest vegetation. T. pulchra trees are 6– 12 m in height with stem diameters that can reach 20 cm. It is a pioneer heliophytic species and can be the dominant species in communities according to environmental conditions. It is very abundant throughout the study area, even in highly polluted areas (Leita˜o Filho et al., 1993). Because of this, it can be considered a good representative of the ecosystem and a suitable species to be studied under field conditions in the region, thus improving the transferability of results to the natural vegetation and facilitating the assessment of the pollution risk for the ecosystem. Saplings of T. pulchra (ca. 30 cm high) were obtained from a nursery and planted in 3-L plastic pots containing a standardized substrate (Eucatex Plantmax (Pinus barks):vermiculite 3:1 v/v) and grown in a greenhouse. Nylon wicks guaranteed the water supply during cultivation and later field exposure. Plastic boxes served as water reservoirs (Klumpp et al, 1994). Hoagland solution was used for fertilization. In the field, pots were kept under shading material (50% reduction in sunlight), which protected the plants from excessive radiation. Two consecutive exposure experiments were performed. Each experiment consisted of exposing six T. pulchra saplings in each of the three sites for 12 weeks, from April to July and from July to September 1998. These months correspond to the worst air quality periods in Cubata˜o, i.e., when pollution dispersion is most difficult (CETESB, 1999). Tables 1 and 2 present the air quality data observed during the development of the present study. While particulate materials and SO2 at the VM site, and SO2, O3, and NOx at the CM site are continuously monitored by CETESB, gaseous fluorides and ammonia were measured at the VM site only during the field campaigns. To measure the studied parameters, all the plants that were exposed on the polluted sites (CM and VM) were taken to the control area (VP) to guarantee standardization of the environmental conditions during the measuring phase. Net carbon dioxide assimilation rate per unit leaf area per unit time at light saturation (Asat ), stomatal conductance to water vapor (gs ) and transpiration rate (E) were measured in the field from 8:30 to 10:30 AM, using a portable infrared gas analyzer system

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Table 1 Air concentrations of SO2, gaseous fluorides, ammonia, and particulate material (mg/m3) during the study period at the Mogi Valley Site, Cubata˜o, Brazil (CETESB, 1999) SO2

May Jun Jul Aug Sept Oct a

Gaseous fluorides


Particulate material

Monthly mean

1-day max.

1-h max.

Monthly mean

1-day max.

Montly mean

1-day max.

Monthly mean

1-day max.

37 41 40 28 19 20

82 81 96 75 67 72

287 370 291 280 206 205

—a —a 1.07 1.63 1.37 —a

—a —a 1.97 3.58 2.29 —a

—a —a —a 32 33 32

—a —a —a 41 86 43

114 109 122 104 86 66

250 175 255 201 207 167

Not determined.

Table 2 Air concentrations of O3, SO2, particulate material (mg/m3), and NOx (ppb) during the study period at the Caminho do Mar Site, Cubata˜o, Brazil (CETESB, 1999) O3

May Jun Jul Aug Sept Oct



Particulate material

Mean of daily 1-h max.

1-h max.

Monthly mean

1-day max.

Monthly mean.

1-day max.

1-h max.

Monthly mean

1-day max

69 68 100 80 78 73

129 171 289 227 177 205

138 136 134 97 87 66

214 211 219 166 135 160

35 28 18 11 12 10

89 63 38 31 44 36

302 242 159 159 154 112

45 46 44 42 37 32

125 216 81 76 82 68

(ADC, LCA-4, UK). It was connected to an 11-cm2 leaf chamber (PCL-N) at saturating photosynthetic photon flux density (900 mmol/m2 s) the value of which was found by photosynthesis light response curves at ambient CO2 concentrations with leaf temperatures from 251C to 271C, and relative humidity ca. 55%. For each exposure experiment, these parameters were measured in six plants per area, in leaves whose limbs had recently been fully expanded. After the field measurements, the following parameters were also determined: presence of visible injury (necrosis or chlorosis); diameter and height increases; biomass of leaves, stems, and roots; leaf area; and the leaf concentration of ascorbate (Keller and Schwager, 1977), chlorophylls a and b (Barnes et al., 1992), nitrogen, sulfur (Zagatto et al., 1981) and fluoride (AOAC, 1975). All chemical determinations were established with n ¼ 6 and 2 replications per plant. Results were statistically compared through analysis of variance (ANOVA). When ANOVA showed significant differences (Po0:05), Bonferroni’s multiple comparison method was performed (Neter et al., 1996).

3. Results Asat was significantly reduced in the plants exposed in VM, during both exposure periods, amounting to 52% of the control area in July and 57% in October

(5.8071.02 in VM, and 10.7870.86 mmol/m2/s in control) (Tables 3 and 4). The plants in CM did not significantly differ from those in the control group (8.8870.81 mmol/m2/s). E and gs rates remained unaffected on both exposure sites (gs was 0.1670.02, 0.1870.03 and 0.1570.02 mol/m2/s and E was 1.8270.14, 1.9370.17, and 1.6970.16 mmol/m2/s, in control, CM, and VM, respectively). Leaf concentrations of chlorophyll a decreased in plants exposed to the CM environment during both exposure times and remained unchanged in VM. Leaf concentrations of chlorophyll b were significantly lower in plants from both CM and VM sites in the first exposure time and in plants from CM during the second exposure time. Changes in the chlorophyll a/b ratio were observed at both polluted sites but only during the first exposure period (Tables 3 and 4). Visible foliar injuries were found in plants from both CM and VM sites. In VM, in both exposure times, all plants presented damages. In CM, 4–6 plants and all plants were damaged in the first and second exposures, respectively. Such injuries include necroses, mainly intravenal, of consistent brown color and irregular shape, which were more intense in the second exposure period at both sites (Table 4). During both experiments all growth parameters were more severely reduced in the saplings exposed to the two polluted sites than in the control saplings. For example, leaf biomass on the VM site corresponded to 24% of the

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Table 3 Carbon assimilation (Asat ), stomatal conductance (gs ), and transpiration rates (E), and chlorophyll a and b contents (means7SE for two exposition periods) in leaves of T. pulchra after exposure on the study sites, Cubata˜o

VP (control) CM VM

Asat (mmol/m2/s)

gs (mol/m2/s)

E (mmol/m2/s)

Chlorophyll a (mg/g DW)

Chlorophyll b (mg/g DW)

10.7870.42 8.8870.39 5.8070.51

0.1670.02 0.1870.03 0.1570.02

1.8270.07 1.9370.09 1.6970.08

2.2670.07 1.8470.07 2.0470.08

0.6870.06 0.5070.02 0.5670.03

Table 4 Gas exchange and chlorophyll concentrations (% of control, mean7SE, n ¼ 6) in T. pulchra saplings leaves after exposure on the study sites, Cubata˜o

Asat gs E Chlorophyll a Chlorophyll b Chlorophyll a/b

Table 7 Concentrations of ascorbate, fluorine, nitrogen, and sulfur (% of control, mean7SE, n ¼ 6) in leaves of T. pulchra saplings after exposure on the study sites, Cubata˜o

First Exposure

Second exposure

First exposure









7778 99714 10278 7977* 6976* 11575*

5277* 92711 9278 8377 6777* 11671*

92711 120711 11575 8275* 8177* 9973

5779* 9477 9577 10075 9778 9672

5974* 120710 13179* 127711*

4376* 919751*,** 17075*,** 12278*

7075* 127711 11673* 10873*

5677* 12837115*,** 15078*,** 11074*

Ascorbate Fluorine Nitrogen Sulfur

Second exposure

*Significantly different from the control (Po0:05) **Significantly different from the other polluted site (Po0:05).

*Significantly different from the control (Po0:05).

First exposure

Second exposure

found in exposed plants on the control site. Fluorine presented the highest accumulation rates: in VM, it amounted to 1283% of the control site value found for the second exposure time (Table 7). N and S leaf concentrations were also significantly higher in plants exposed in polluted areas (Table 7).

070 1174 55711

070 7274 73719

4. Discussion

Table 5 Visible foliar injury of saplings of T. pulchra (mean7SE, n ¼ 6) after exposure on the study sites, Cubata˜o Study site

Control CM VM

% Damaged leaves per sapling

Table 6 Growth parameters (% of control, mean7SE, n ¼ 6) in saplings of T. pulchra after exposure on the study sites, Cubata˜o

Height increase Diameter increase Leaf biomass Root biomass Whole plant biomass Leaf area

First exposure

Second exposure





64713* 6376* 5278* 4774* 5276* 3877*

74712* 38710* 2476*,** 4576* 3674*,** 5778*

49710* 6375* 52710* 5677* 5378* 2573*,**

2674* 43715* 57712* 30710* 3879*,** 5779*

*Significantly different from the control site (Po0:05).

values obtained for plants from the control site in the first exposure experiment, and leaf area on the CM site was 25% of the control site in the second experiment (Table 5). Ascorbate leaf concentrations were also lower in saplings exposed on the CM and VM sites (Table 6). Leaf concentrations of phytotoxic or potentially phytotoxic elements increased as compared to those

Compared to values registered in plants from the control site, Asat was lower in plants exposed on the VM site (Po0:05) and remained the same in plants on the CM site. The photosynthesis process is sensitive to air pollution and often decreases in plants on polluted locations, whether native or introduced (Gratani et al., 2000; Zhang et al., 2001). In these plants, photosynthesis reduction results from damages related to stomatal movements, light trap, or CO2 fixation. As in this study no differences in stomatal conductance rates were found; the Asat reduction observed might have been caused by changes in light trap or CO2 fixation. Renaud et al. (1997) and Shan (1998) also observed reductions in Asat and not in gs : Stomatal closure seems to be an indirect effect of plant exposure to pollutants, and it maintains the internal concentration of CO2 once carboxylation has been affected. According to Pell et al. (1994), of all the photosynthesis processes, carboxylation is the most sensitive to O3 effects. Carboxylation reduction can be associated with a decrease in the activity and synthesis of ribulose-1,5biphosphate carboxylase-oxygenase, which seems to be the case in this study.


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The plants in CM presented significant reductions in all the growth parameters measured, even though Asat was similar to the results observed in plants from the control site. Edwards et al. (1994) and Robinson and Britz (2000) also observed growth reductions without a concomitant Asat decrease in plants exposed to pollutants. Probably most of the carbon assimilated by the CM plants could not be used in phytomass production. Consequently, although net photosynthesis values expressed per unit of area were similar to those found in the control group, when the whole plant foliage was taken into account, carbon assimilation was lower in plants on the CM site, given the smaller leaf area. The amount of carbon assimilated by plants cannot be directly incorporated into biomass, since part of it will be used in the respiratory process. According to Mooney and Winner (1988), stress causes an increase in the rate of respiratory metabolism for performing functions such as the repair of damaged cellular constituents or the increased production of some compounds (ATP and reductants) required for detoxification or defense. This is a common response to chronic air pollution stress, since at the same time energy and carbon skeleton production for growth may be reduced (Amthor and McCree, 1990). Plants exposed on the CM site might also have invested more carbon in maintenance and detoxification functions, thus decreasing the carbon available for growth. The reductions in the contents of ascorbate are indications of this. Chlorophyll a concentrations did not decrease in plants exposed on the VM site, probably due to higher foliar nitrogen accumulation rates in plants exposed to several NOx and NH4 emission sources in the Mogi Valley. The highest N emissions may have favored chlorophyll synthesis, partially compensating the degradation induced by other pollutants, as Shan (1998) also observed. According to Maandre and Tuulmets (1997), chlorophyll a would be more sensitive to air pollution than chlorophyll b, and one can conclude that the increase in the chlorophyll a/b ratio found in VM and CM plants during the first exposure time would be a good indication of metabolic changes. The lower leaf ascorbate concentrations on both polluted sites during both exposure times provide evidence that the plants were submitted to oxidative stress. Ascorbate is a key antioxidant in the neutralization of active oxygen species and it can directly eliminate several of them, such as singlet oxygen, superoxide, and the hydroxyl radical (Conklin, 2001). Reduction in the content would reveal its use in detoxification (Klumpp et al., 2000). Moreover, according to Robinson and Britz (2000), glucose plays a major role in the maintenance of ascorbate levels, since it is the galactose precursor, which, in turn, is the ascorbate precursor. Therefore, photosynthesis inhibition in plants exposed

on the VM site might also have contributed to the lower ascorbate levels in plants kept in the location. The high foliar accumulation of fluorine in T. pulchra from the VM site demonstrates that, despite reduction, fluoride emissions from phosphate fertilizer plants in VM are still at extremely high levels and may be related to the reduction of Asat observed in plants exposed at this site. Fluoride emissions are probably the major cause of degradation in the region’s Atlantic forest. The significantly higher concentrations of S and N in plants from the polluted sites also indicated that issues related to S and N emissions still remain unsolved. 5. Conclusions The results reconfirm previous studies (Domingos et al., 1998; Klumpp et al., 2000) that demonstrated that air pollution in Cubata˜o still reaches phytotoxic levels. Moreover, the study periods seem to have been particularly unfavorable for plant development, since previous studies revealed neither the growth reductions observed in plants exposed on the VM site (Klumpp et al., 2000) nor the presence of visible foliar injuries. Reductions in growth parameters demonstrate that air quality in the areas near the petrochemical plants (CM site) is also compromised and has led to a severe delay in the development of T. pulchra in the zone. Processes related to Asat seem to be less sensitive to air pollution exposure than those related to growth; the former remained unchanged in plants exposed on the CM site. The differences between Asat and growth responses allow one to discriminate sites with regard to the pollution phytotoxicity level, revealing a clear gradient among the study sites, with VM being the worst site with respect to air quality. VM plants presented the most intense reactions to air pollution in terms of the presence of injuries, reductions in Asat and leaf biomass, and increased whole plant and foliar F and N accumulation. One of the causes of these intense effects must surely be the extremely high F accumulation detected in plants exposed on the VM site. Based on the results of this study it is possible to conclude that the accumulation of phytotoxic and potentially phytotoxic elements and the metabolic changes in saplings of T. pulchra, a species considered resistant to air pollution, provide evidence that, in the Cubata˜o region, air pollution remains a stress and risk factor for this species and for the vegetation of the Atlantic forest. Acknowledgments The authors thank Shoey Kanashiro for the guidelines in phytosanitary care and FAPESP for the financial support provided (Proc. 97/12163-7).

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References Amthor, J.S., McCree, K.J., 1990. Carbon balance of stressed plants: a conceptual model for integrating research results. In: Alscher, R.G., Cumming, J.R. (Eds.), Stress Responses in Plants: Adaptation and Acclimation Mechanism. Wiley-Liss, New York, pp. 1–15. AOAC (Association of Official Analytical Chemists), 1975. Fluoride potentiometric method—official first action. J. Assoc. Official Anal. Chem. 58, 384–385. Barnes, J., Balanguer, L., Manrique, E., Elvira, S., Davison, A.W., 1992. A reappraisal of the use of DMSO for the extraction and determination of chlorophylls a and b in lichens and higher plants. Environ. Exp. Bot. 32, 85–100. CETESB (Companhia de Tecnologia e Saneamento Ambiental), 1999. Relato´rio Anual da Qualidade do Ar no Estado de Sa˜o Paulo—1998. CETESB, Sa˜o Paulo. Conklin, P.L., 2001. Recent advances in the role and biosynthesis of ascorbic acid in plants. Plant Cell Environ. 24, 383–394. Domingos, M., Klumpp, A., Klumpp, G., 1998. Air pollution impact on the Atlantic Forest at the Cubata˜o region, Brazil. Cieˆn. Cult. 50, 230–236. Edwards, G.S., Wullschleger, S.D., Kelly, J.M., 1994. Growth and physiology of northern red oak: preliminary comparison of mature tree and seedling responses to ozone. Environ. Pollut. 83, 215–221. Furlan, C.M., Salatino, A., Domingos, M., 1999. Leaf contents of nitrogen and phenolic compounds and their bearing with herbivore damage to Tibouchina pulchra Cogn, (Melastomataceae), under the influence of air pollutants from industries of Cubata˜o, Sa˜o Paulo. Revta. Bras. Bot. 22, 317–323. Gratani, L., Crescente, M.F., Petruzzi, M., 2000. Relationship between leaf life span and photosynthetic activity of Quercus ilex in polluted urban areas (Rome). Environ. Pollut. 110, 19–28. Jaeschke, W., 1997. Chemistry module. In: Klockow, D., Targa, H.T., Vautz, W. (Eds.), Air Pollution and Vegetation Damage in the Tropics—The Serra do Mar as an Example—Final Report 1990– 1996. GKSS-Forschungszentrum Geesthacht GmbH, Geesthacht, pp. II.1–II.77. Keller, T., Schwager, H., 1977. Air pollution and ascorbic acid. Eur. J. Forest Pathol. 7, 338–350. Klumpp, A., Domingos, M., Klumpp, G., 1996a. Assessment of the vegetation risk by fluoride emissions from fertilizers industries at Cubata˜o, Brazil. Sci. Total. Environ. 192, 219–228. Klumpp, A., Domingos, M., Klumpp, G., Guderian, R., 1997. Vegetation module. In: Klockow, D., Targa, H.T., Vautz, W. (Eds.), Air Pollution and Vegetation Damage in the Tropics— The Serra do Mar as an Example—Final Report 1990–1996, GKSS-Forschungszentrum Geesthacht GmbH, Geesthacht, pp. IV.1–IV.64.


Klumpp, A., Domingos, M., Moraes, R.M., Klumpp, G., 1998. Effects of complex air pollution on tree species of the Atlantic rain Forest near Cubata˜o, Brazil. Chemosphere 36, 989–994. Klumpp, A., Klumpp, G., Domingos, M., 1994. Plants as bioindicators of air pollution at the Serra do Mar near the industrial complex of Cubata˜o, Brazil. Environ. Pollut. 85, 109–116. Klumpp, A., Klumpp, G., Domingos, M., Silva, M.D., 1996b. Fluoride impact on native tree species of the Atlantic Forest near Cubata˜o, Brazil. Water Air Soil Pollut. 87, 57–71. Klumpp, G., Furlan, C.M., Domingos, M., Klumpp, A., 2000. Responses of stress indicators and growth parameters of Tibouchina pulchra Cogn. exposed to air and soil pollution near the industrial complex of Cubata˜o, Brazil. Sci. Total Environ. 246, 79–91. Leita˜o Filho, H.F., Pagano, S.N., Cesar, O., Timoni, J.L., Rueda, J. J., 1993. Ecologia da Mata Atlaˆntica em Cubata˜o. Editora da Universidade Estadual Paulista, Rio Claro. Maandre, M., Tuulmets, L., 1997. Pigment changes in Norway spruce induced by dust pollution. Water Air Soil Pollut. 94, 247–258. Mooney, H.A., Winner, W.E., 1988. Carbon gain, allocation, and growth as affected by atmospheric pollutants. In: Schulte-Hostede, S., Darrall, N.M., Blank, L.W., Wellburn, A.R. (Eds.), Air Pollution and Plant Metabolism. Elsevier, London, pp. 272–287. Neter, J., Kutner, M.H., Nachtscheim, C.J., Wasserman, W., 1996. Applied Linear Statistical Models. Times Mirror Higher Education Group, New York. Pell, E.J., Eckart, N.A., Glick, R.E., 1994. Biochemical and molecular basis for impairment of photosynthesis potential. Photos. Res. 39, 453–462. Renaud, J.P., Laitat, E., Maufette, Y., Allard, G., 1997. Photoassimilate allocation and photosynthetic and biochemical characteristics of two alfalfa (Medicago sativa L.) cultivars of different ozone sensitivities. Can. J. Bot. 76, 281–289. Robinson, J.M., Britz, S.J., 2000. Tolerance of a field grown soybean cultivar to elevated ozone level is concurrent with higher leaflet ascorbic acid level, higher ascorbate–dehydroascorbate redox status, and long term photosynthetic productivity. Photos. Res. 64, 77–87. Shan, Y., 1998. Effects of simulated acid rain on Pinus densiflora: inhibition of net photosynthesis by the pheophytization of chorophyll. Water Air Soil Pollut. 103, 121–127. Zagatto, E.A.G., Jacintho, A.O., Reis, B.F., Krug, F.J., Bergamin Filho, H., Pessenda, L.C.R., Mortatti, J., Gine´, M.F., 1981. Manual de Ana´lise de Plantas e A´gua Empregando Sistemas de Injec-a˜o em Fluxo. CENA/USP, Piracicaba. Zhang, J., Ferdinand, J.A., Vanderheyden, D.J., Skelly, J.M., Innes, J.L., 2001. Variation of gas exchange within native plant species of Switzerland and relationships with ozone injury: An open-top experiment. Environ. Pollut. 113, 177–185.