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n-Alkane distribution of leaves of Psidium guajava exposed to industrial air pollutants
Environmental and Experimental Botany 58 (2006) 100–105
n-Alkane distribution of leaves of Psidium guajava exposed to industrial air pollutants Cl´audia M. Furlan a,∗ , D´eborah Y.A.C. Santos a , Antonio Salatino a , Marisa Domingos b a
Departamento de Botˆanica, Instituto de Biociˆencias, Universidade de S˜ao Paulo, CP 11461, 05422-970 S˜ao Paulo, SP, Brazil b Se¸ ca˜ o de Ecologia, Instituto de Botˆanica, SMA, CP 4005, 01061-970 S˜ao Paulo, Brazil Received 3 March 2005; received in revised form 4 May 2005; accepted 20 June 2005
Abstract The distribution of n-alkanes of foliar epicuticular waxes of Psidium guajava (Myrtaceae) exposed to air pollutants was determined by GCEIMS. Saplings were exposed for two periods of one year (July 2000–June 2001 and December 2000–November 2001) in two experimental sites: Pil˜oes River Valley (PV), where the contamination of air pollutants is relatively low, and Mogi River Valley (MV), which is severely affected by pollutants released by chemical, fertilizer, ceramic, iron and steel industries. On average, total leaf waxes were higher for plants that were exposed initially in the rainy season (December). Leaf waxes showed different n-alkane composition comparing both sites, especially during the first period of exposure (July 2000–June 2001). The main n-alkane waxes in both areas were C25 , C26 and C27 . However, in both experimental periods, an increase of the relative amounts of homologous of shorter carbon chains (C18 –C23 ) under pollutants influence was observed. While the total percents of such homologues were on average 9.5 and 10.2 at PV for the first and second experimental periods, the observed percents at MV were on average 15.6 and 13.7, respectively. The results of the present work are in agreement with previous findings that air pollutants affect the synthesis of surface waxes, leading to alterations of the distribution of wax constituents. © 2005 Elsevier B.V. All rights reserved. Keywords: n-Alkanes; Psidium guajava; Myrtaceae; Air pollution; Cubat˜ao; Epicuticular waxes
1. Introduction Epicuticular waxes of higher plants provide a physiochemical barrier that reduces non-stomatal water loss and may control leaf temperature, photoprotection, frost hardness, penetration of foliar applied compounds and acts as a protection against pest attacks (insects, fungi and bacteria) (Shepherd et al., 1995; Barnes and Cardoso-Vilhena, 1996; Bally, 1999; Riederer and Schreiber, 2001). The epicuticular waxes consist of complex mixtures of long chain aliphatic and cyclic components, including hydrocarbons, primary and secondary alcohols, aldehydes, ketones, esters, fatty acids and triterpenoids (Shepherd et al., 1995; Santier and Chamel, 1998; Kroumova and Wagner, 1999; Jetter and Sch¨affer, 2001). In higher plants, hydrocarbons are ubiquitous in foliar waxes, comprising mainly a wide range (C15 –C38 ) of n∗
Corresponding author. Tel.: +55 11 3091 7532; fax: +55 11 3091 7547. E-mail address: [email protected] (C.M. Furlan).
0098-8472/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.envexpbot.2005.06.020
alkane homologues with the predominance of compounds with odd number of carbons atoms (Bianchi, 1995; Reddy et al., 2000). Chain length distributions of wax components have been reported to change with variation in environmental conditions such as temperature, light intensity and air pollution (Poborski, 1988; Kerfourn and Garrec, 1992; Percy et al., 1994; Shepherd et al., 1995). The epicuticular waxes of plants growing at polluted sites are the first foliar components to be attacked by both gaseous and particulate pollution. The formation of epicuticular waxes can be restrained and their composition and distribution changed. The ultimate result may be a corrosive damage of the leaf surfaces and/or a mechanical blockage of stomata (Cape and Percy, 1998; Bytnerowicz et al., 1998; G¨unthardt-Goerg et al., 2000). In the present paper, the wax morphology and the relative abundance of n-alkane homologues of foliar waxes of saplings of Psidium guajava L. (Myrtaceae) were determined, exposed over one year to a highly polluted and a
C.M. Furlan et al. / Environmental and Experimental Botany 58 (2006) 100–105
non-polluted area around the industrial complex of Cubat˜ao, S˜ao Paulo. Two annual exposure experiments were carried out, one starting in the winter, the highest polluted period of the year, and another starting in the summer, when air pollutant concentrations are generally lower. This study aims to evaluate the extent of alterations caused by air pollutants to one of the major constituents of the epicuticular wax of P. guajava, a tropical tree that has been used as bioindicator of air pollution.
2. Material and methods 2.1. Experimental areas
Table 1 Gaseous air pollutants concentration (ppm) at the PV (non-polluted site) and MV (polluted site) Date
SO2 (ppm)a PV
MV
PV
MV
PV
MV
March 2001 April 2001 May 2001 June 2001 July 2001 August 2001 September 2001 October 2001 November 2001 December 2001
<0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05
0.12 – 0.10 0.05 0.10 0.70 0.30 0.10 0.20 0.15
<0.10 <0.10 <0.10 <0.10 <0.10 0.10 <0.10 <0.10 <0.10 <0.10
0.50 0.70 0.70 0.50 0.50 1.00 0.70 1.50 1.50 1.50
<0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50
0.50 0.50 0.50 0.25 0.50 1.00 0.50 0.50 0.50 0.50
a
The studied areas are located near the industrial complex of Cubat˜ao in the state of S˜ao Paulo, SE-Brazil (23◦ 45 –23◦ 55 S, 46◦ 15 –46◦ 30 W), which lies between the city of Santos and the slopes of Serra do Mar. The topographic location of the industrial complex and unfavorable meteorological conditions hinders the dispersion of pollutants. The climate is tropical with annual mean temperatures of 23 ◦ C, with no dry season, high humidity and annual precipitation of 2600 mm at the coastal plain. This is one of the most important industrial complexes of South America, housing 11 petrochemical/chemical industries, 7 fertilizer industries, 1 steel work and 1 cement factory, totaling 206 emission sources of gas pollutants (CETESB, 2002). Highly polluted and non-polluted sites, respectively, Mogi River Valley (MV) and Pil˜oes River Valley (PV) were selected to install the exposure system, based on previous studies that demonstrated differences between the two sites only with regard to the local air contamination. Similar altitudes (around 50 a.s.l.) and meteorological conditions prevail at both sites: they differ with regard to the nature of the local air pollutants (Klumpp et al., 2000; Fiedler and Massambani, 2001; Moraes et al., 2002). MV is in close proximity to the core of the industrial complex, downwind of the industrial complex, and characterized by severe vegetation damage. High contamination of particulate matter, fluorides, sulfur and nitrogen compounds are assumed to predominate at MV (Jaeschke, 2001). Concentrations of 184 g m−3 , 35 g m−3 and 29 g m−3 of total particulate matter, nitrogen oxides and sulfur dioxide, respectively, were reported by the local monitoring station in 2000 (CETESB, 2002). PV site is southwest of the industrial complex and was assumed as the reference area. Low concentration of air pollutants stemming mainly from vehicles characterizes PV. Geographic barriers and location out of reach by the landto-sea breeze determine that the air pollutants emitted by Cubat˜ao industrial complex contaminate only slightly the areas embraced by PV (Jaeschke, 2001). The concentrations of SO2 , fluorides and NOx , the most abundant pollutants at the MV site, were measured at both experimental sites, from March to November 2001,
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b c
Fluorides (ppm)b
NOx (ppm)c
1 ppm SO2 = 2.67 mg SO2 m−3 . 1 ppm fluoride = 1.58 mg HF m−3 . 1 ppm NO2 = 1.92 mg NO2 m−3 .
by monthly discontinuous samplings with a manual pump (Dr¨agger-Accuro) by a colorimetric approach (Table 1). The accumulated precipitation, the number of unfavorable days for pollutant dispersion and the concentrations of sulfur dioxide (SO2 ), nitrogen dioxide (NO2 ) and particulate matter (MP10) are shown in Table 2 in order to characterize the two periods of experiment (July 2000–June 2001 and December 2000–November 2001) at MV site. It is important to point out that PV is not monitored by the State Agency of Pollution Monitoring (CETESB), since the region is considered non-polluted. 2.2. Plant exposure Saplings of P. guajava L. (about 30 cm high) were obtained from a nursery and planted in plastic pots containing uniform soil. In the field, pots were kept under a shading protection that promoted a 50% reduction of incident sunlight. Thirty-six potted saplings were maintained at both sites during two periods of one year (July 2000–June 2001 and December 2000–November 2001). After 12 months of exposure, undamaged and fully expanded leaves of the fourth node of each sapling were collected for wax chemical and morphological analyses, corresponding to a leaf that expanded after transport to the study sites. The total leaf area used for wax extractions was estimated by scanning leaves using the Scion Image Beta 4.02 program (Scion Corporation, USA). 2.3. Wax morphology Leaves were dried at room temperature. Three inter-venal regions were selected for three saplings of each site to be analyzed. The samples were placed on aluminum-stubs using a double-sided adhesive tape. The specimens were sputter coated with a thin gold-layer and were investigated by scanning electron microscopy (SEM) (ZEISS DSM 940, Germany). To visualize the wax morphology, abaxial and adaxial
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Table 2 Accumulated precipitation (PPT, mm), unfavorable days for pollutant dispersion (UDD) and mean and maximum concentration (g m−3 ) of nitrogen dioxide (NO2 ), sulfur dioxide (SO2 ) and particulate matter (PM10) at Cubat˜ao during periods of exposure PPTa
UDDb
NO2 b Mean
Period I Winter Spring Summer Autumn Total accumulated Annual mean Period II Summer Autumn Winter Spring Total accumulated Annual mean
167.0 692.2 742.0 520.0
31 2 0 13
59.7 49.7 49.0 60.3
2091.2 174.3
46 –
593.0 53.9
742.0 431.1 259.5 569.4
0 4 30 3
49.0 60.3 77.0 57.7
2002.0 166.8
37 –
665.0 59.5
SO2 b Maximum 110.0 94.0 88.0 113.0
Mean 43.3 23.3 20.7 27.7
PM10b Maximum 114.0 49.0 59.0 77.0
328.0 27.3 88.0 113.0 176.0 101.0
20.7 27.7 35.3 24.0
Mean 183.3 162.3 212.9 197.9
Maximum 347.0 320.0 368.0 407.0
2279.6 190.0 59.0 77.0 112.0 96.0
323.0 26.9
212.9 197.9 244.1 231.1
368.0 407.0 587.0 306.0
2658.2 221.5
Period I: July 2000–June 2001; Period II: December 2000–November 2001. a Data from Empresa Metropolitana de Aguas ´ e Energia (EMAE). b Data from Companhia de Tecnologia e Saneamento Ambiental (CETESB).
leaf surfaces were observed in secondary electron mode, at an acceleration voltage of 10 keV and magnification of 1000×. The wax morphology of the abaxial and adaxial leaf surfaces was investigated before and after the wax chloroform extraction, described in the next section. 2.4. Wax analysis Foliar waxes of all saplings were extracted with chloroform by three consecutive immersions of 10 s each. The extracts were filtered through chloroform-washed filter paper and the solvent was evaporated under reduced pressure. The extracted waxes were weighted and the alkane fraction was separated by column chromatography (CC) using aluminum oxide and n-hexane. Alkanes were analyzed by gas chromatography and mass spectroscopy, using a capillary column DB-5HT HP (30 m, 0.32 mm), helium at a flow rate of 1 mL min−1 and electronic impact method at 70 eV. The temperatures of the injector and the detector were 300 ◦ C. The temperature of the column ranged between 140 ◦ C (1 min) and 300 ◦ C at 10 ◦ C min−1 . The temperatures of the MS source and quadruple were, respectively, 250 ◦ C and 100 ◦ C. The identification of the individual alkanes was achieved by the corresponding mass spectra and comparison of retention times with those of n-alkane standards. 2.5. Statistical analysis The percentage data were transformed into the corresponding square roots for each value. The raised data were statistically treated using ANOVA. The procedure of pairwise comparison (Bonferroni’s test) was used when ANOVA revealed significant differences between sites and year periods.
3. Results and discussion The morphology of abaxial and adaxial leaf surfaces of P. guajava after 12 months of exposure to air pollutants emitted by the industrial complex of Cubat˜ao is shown in Fig. 1. The morphology of leaf surfaces before and after chloroform extraction of total wax showed no differences, suggesting that the leaf epidermal cells in P. guajava are covered by a thin wax layer. Total wax contents (TWC) were not significantly different in leaves of saplings exposed at both sites (Table 3). On the other hand, higher quantities of wax are noted for saplings in both sites comparing the periods December 2000–November 2001 and July 2000–June 2001. These results suggest a stronger climatic influence on wax production than a pollution effect. Faini et al. (1999) studying seasonal changes in chemical composition of epicuticular waxes of leaves of Baccharis linearis observed higher production of epicuticular waxes in summer, when drought and solar radiation are highest. Higher production of epicuticular waxes in summer may be advantageous inasmuch as the waxes may act as a physical barrier to prevent water permeation and dehydration of the leaves, in addition to filtering and reflecting solar radiation. According to Riederer and Schreiber (2001), the cuticle is the major barrier against uncontrolled water loss from leaves, fruits and other parts of higher plants. It is known that the water permeability of the plant cuticle is determined by wax composition and ultrastructure. Studies involving extraction of total foliar waxes showed an increase of the cuticle permeability to sulfur dioxide, hydrogen fluoride, carbon dioxide and herbicides (Poborski, 1988; Santier and Chamel, 1998). Plants under water stress have been found to have an increase of the waxes total amount (Poborski, 1988; Bondada et al., 1996). In addition, leaves of
C.M. Furlan et al. / Environmental and Experimental Botany 58 (2006) 100–105
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Fig. 1. Scanning electron microscopy of leaves of Psidium guajava exposed for 12 months at two areas: (A) abaxial and (B) adaxial leaf surfaces of saplings exposed at control site (PV), respectively; (C) abaxial and (D) adaxial leaf surfaces of saplings exposed at polluted site (MV), respectively; (E) adaxial leaf surface after wax extraction. Bars: 10 m. Note abundant deposits of particulate pollutants in (D).
heavily polluted areas exhibit enhanced water loss simultaneously with changes in the wax composition and ultrastructural alterations (Cape and Percy, 1998; Bytnerowicz et al., 1998; Viskari et al., 2000). It was noted that not only the amounts of wax are important to protect the leaf tissues against water
loss, but also their chemical composition, in especially long chain aldehydes, free primary alcohols and other non-polar substances (Poborski, 1988; Bondada et al., 1996). The n-alkane wax homologues of P. guajava after 12 months of exposure in both areas were predominantly C25 ,
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Table 3 Relative abundance (%) of n-alkanes and total wax content (TWC) of leaves of Psidium guajava saplings (N = 36) exposed for two periods of 12 months at polluted (MV) and non-polluted (PV) sites Chain length
July 2000– June 2001
December 2000– November 2001
PV
MV
PV
MV
C19 C20 C21 C22 C23 C24 C25 C26 C27 C28 C29 C30 C31 C19 –C23 C24 –C29 Codd Ceven Even/odd C19 –C23 /C24 –C29
0.16a 1.54a 0.37a 3.41a 4.48a 9.70a 20.56a 20.06a 22.96a 5.53a 8.98a 0.97a 1.28a 9.96a 90.04a 58.79a 41.21a 0.70a 0.11a
0.28a 2.64a 1.25b 4.77b 7.35b 11.38a 18.38a 16.68b 20.98b 7.18b 7.16a 0.98b 0.99a 16.28b 83.72b 56.38b 43.62a 0.77b 0.19b
0.00a 0.81a 0.00a 5.44a 3.52a 14.19a 16.29a 23.59a 20.99a 6.43a 7.30a 1.34a 0.10a 9.77a 90.23a 48.20a 51.80a 1.07a 0.11a
1.09a 0.71a 0.50a 8.57a 6.33b 13.44a 17.34a 20.14b 18.44a 7.30a 4.65a 1.38a 0.07a 17.22a 82.78a 48.44a 51.56a 1.06a 0.21a
TWC (g cm−2 )
34.6b
40.1b
73.8a
88.6a
Values followed by different letters (a and b) correspond to different means (P ≤ 0.05) between PV and MV at the same exposure period. For TWC, values followed by different letters (a and b) correspond, for the same site, to different means (P ≤ 0.05) between July 2000–June 2001 and December 2000–November 2001.
C26 and C27 (Table 3). However, an increase of the relative amounts of homologues with shorter carbon chains (C19 –C23 ) and a decrease of the relative amounts of homologues with longer carbon chains (C24 –C31 ) were noted in the foliar waxes of saplings under pollutants influence (Table 3) exposed in the period starting in the winter (July). Although not significant, the values observed for the second period of exposure showed a similar trend toward higher amounts of homologues with shorter chains. Even/odd and short/long carbon chains ratios of leaf waxes increased in MV in the first period of exposure (July 2000–June 2001; Table 1). The climatic conditions of the first months of exposure seem to be important to establishing the intensity of plant responses to air pollution. In the experiment started in the winter, the plants grew over the first six months under drier atmosphere and more unfavorable conditions for pollutant dispersion, reaching the rainy summer and autumn periods only in the last six months. On the other hand, the second period of exposure (December 2000–November 2001) corresponds to summer and autumn for the first six months and to winter and spring for the final months. The results suggest that harsher growth conditions (low moisture and more pollutants) in the first months of exposure are more effective to alter wax composition than the milder conditions prevalent in the wetter seasons.
Turunen et al. (1997) studied the response of epicuticular wax of Pinus sylvestris needles to dry and wet deposited sulfur and heavy metals found that sulfur deposition plays a more important role in needle surface deterioration than heavy metals. Sulfur dioxide and acid rain have been found to be more deleterious to needles surfaces than ozone (Cape et al., 1995). Significant differences in n-alkanes composition occurred only during the first exposure period, when atmospheric sulfur dioxide concentration was highest (Table 3). Kerfourn and Garrec (1992) studied Hedera helix and Picea abies exposed to acid and ozone found higher amounts of short chain and lower amounts of long chain alkanes in P. abies, as well as increased ratios of even to odd chains for H. helix leaves exposed to acid rain. Barnes et al. (1994) studied Nicotiana tabacum exposed to enhanced UV-B radiation also found shifts of n-alkane composition toward shorter chain lengths. Turunen et al. (1997) found effects on wax chemical composition of P. sylvestris due to the proximity of a smelter industry. Significant site influences were found especially for secondary alcohols, the major constituent of P. sylvestris waxes. The results observed in saplings of P. guajava exposed to air pollutants are similar to those found by Kerfourn and Garrec (1992). The alteration might be caused by the presence of higher concentrations of SO2 and NOx in Cubat˜ao, which contribute to rain acidification (CETESB, 2002). The alterations on alkane composition are particularly interesting because of their relationship with foliar penetration by chemicals, particularly taking into account that they belong to the most hydrophobic class of plant waxes. Long chain alkanes are known to be more resistant to the penetration of polar solutions than shorter homologues, in addition to playing an important role as plant protectors against adverse environmental conditions, such as water stress (Kerfourn and Garrec, 1992; Bianchi, 1995; Bondada et al., 1996). Oliveira et al. (2003) verified that the chemistry of the wax constituents is a factor more important to determine the degree of resistance to evaporation than the thickness of the waxy deposits. The authors verified that n-alkanes and alcoholic triterpenes were more efficient barriers to water loss than hentriacontan16-one (a ketone) and ursolic acid (an acid triterpene). Oliveira and Salatino (2000) investigated the major constituents of foliar waxes of species from caatinga (semi-arid ecosystem in northeast Brazil) and cerrado (savanna ecosystem in central and southeast Brazil). Although n-alkanes and triterpenoids are important constituents of waxes of both habitats, the former substances were found to be more common and abundant in plants from the caatinga. The present results are the first report about effects of air pollutants on n-alkane distributions in leaf waxes of a tropical tree species. A question may be raised at this point whether the observed induced distribution may have reflections on alterations of the efficiency of water retention. Studies combining these approaches are important to integrate the influence of pollution stress and other environmental factors.
C.M. Furlan et al. / Environmental and Experimental Botany 58 (2006) 100–105
Acknowledgements The authors thank FAPESP (Proc. No. 99/06971-9) and CNPq for provision of funds, and Instituto Florestal (N´ucleo Pil˜oes) and Cesari Ltda for allowing the use of the areas for exposure of the saplings.
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n-Alkane distribution of leaves of Psidium guajava exposed to industrial air pollutants Cl´audia M. Furlan a,∗ , D´eborah Y.A.C. Santos a , Antonio Salatino a , Marisa Domingos b a
Departamento de Botˆanica, Instituto de Biociˆencias, Universidade de S˜ao Paulo, CP 11461, 05422-970 S˜ao Paulo, SP, Brazil b Se¸ ca˜ o de Ecologia, Instituto de Botˆanica, SMA, CP 4005, 01061-970 S˜ao Paulo, Brazil Received 3 March 2005; received in revised form 4 May 2005; accepted 20 June 2005
Abstract The distribution of n-alkanes of foliar epicuticular waxes of Psidium guajava (Myrtaceae) exposed to air pollutants was determined by GCEIMS. Saplings were exposed for two periods of one year (July 2000–June 2001 and December 2000–November 2001) in two experimental sites: Pil˜oes River Valley (PV), where the contamination of air pollutants is relatively low, and Mogi River Valley (MV), which is severely affected by pollutants released by chemical, fertilizer, ceramic, iron and steel industries. On average, total leaf waxes were higher for plants that were exposed initially in the rainy season (December). Leaf waxes showed different n-alkane composition comparing both sites, especially during the first period of exposure (July 2000–June 2001). The main n-alkane waxes in both areas were C25 , C26 and C27 . However, in both experimental periods, an increase of the relative amounts of homologous of shorter carbon chains (C18 –C23 ) under pollutants influence was observed. While the total percents of such homologues were on average 9.5 and 10.2 at PV for the first and second experimental periods, the observed percents at MV were on average 15.6 and 13.7, respectively. The results of the present work are in agreement with previous findings that air pollutants affect the synthesis of surface waxes, leading to alterations of the distribution of wax constituents. © 2005 Elsevier B.V. All rights reserved. Keywords: n-Alkanes; Psidium guajava; Myrtaceae; Air pollution; Cubat˜ao; Epicuticular waxes
1. Introduction Epicuticular waxes of higher plants provide a physiochemical barrier that reduces non-stomatal water loss and may control leaf temperature, photoprotection, frost hardness, penetration of foliar applied compounds and acts as a protection against pest attacks (insects, fungi and bacteria) (Shepherd et al., 1995; Barnes and Cardoso-Vilhena, 1996; Bally, 1999; Riederer and Schreiber, 2001). The epicuticular waxes consist of complex mixtures of long chain aliphatic and cyclic components, including hydrocarbons, primary and secondary alcohols, aldehydes, ketones, esters, fatty acids and triterpenoids (Shepherd et al., 1995; Santier and Chamel, 1998; Kroumova and Wagner, 1999; Jetter and Sch¨affer, 2001). In higher plants, hydrocarbons are ubiquitous in foliar waxes, comprising mainly a wide range (C15 –C38 ) of n∗
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alkane homologues with the predominance of compounds with odd number of carbons atoms (Bianchi, 1995; Reddy et al., 2000). Chain length distributions of wax components have been reported to change with variation in environmental conditions such as temperature, light intensity and air pollution (Poborski, 1988; Kerfourn and Garrec, 1992; Percy et al., 1994; Shepherd et al., 1995). The epicuticular waxes of plants growing at polluted sites are the first foliar components to be attacked by both gaseous and particulate pollution. The formation of epicuticular waxes can be restrained and their composition and distribution changed. The ultimate result may be a corrosive damage of the leaf surfaces and/or a mechanical blockage of stomata (Cape and Percy, 1998; Bytnerowicz et al., 1998; G¨unthardt-Goerg et al., 2000). In the present paper, the wax morphology and the relative abundance of n-alkane homologues of foliar waxes of saplings of Psidium guajava L. (Myrtaceae) were determined, exposed over one year to a highly polluted and a
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non-polluted area around the industrial complex of Cubat˜ao, S˜ao Paulo. Two annual exposure experiments were carried out, one starting in the winter, the highest polluted period of the year, and another starting in the summer, when air pollutant concentrations are generally lower. This study aims to evaluate the extent of alterations caused by air pollutants to one of the major constituents of the epicuticular wax of P. guajava, a tropical tree that has been used as bioindicator of air pollution.
2. Material and methods 2.1. Experimental areas
Table 1 Gaseous air pollutants concentration (ppm) at the PV (non-polluted site) and MV (polluted site) Date
SO2 (ppm)a PV
MV
PV
MV
PV
MV
March 2001 April 2001 May 2001 June 2001 July 2001 August 2001 September 2001 October 2001 November 2001 December 2001
<0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05
0.12 – 0.10 0.05 0.10 0.70 0.30 0.10 0.20 0.15
<0.10 <0.10 <0.10 <0.10 <0.10 0.10 <0.10 <0.10 <0.10 <0.10
0.50 0.70 0.70 0.50 0.50 1.00 0.70 1.50 1.50 1.50
<0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50 <0.50
0.50 0.50 0.50 0.25 0.50 1.00 0.50 0.50 0.50 0.50
a
The studied areas are located near the industrial complex of Cubat˜ao in the state of S˜ao Paulo, SE-Brazil (23◦ 45 –23◦ 55 S, 46◦ 15 –46◦ 30 W), which lies between the city of Santos and the slopes of Serra do Mar. The topographic location of the industrial complex and unfavorable meteorological conditions hinders the dispersion of pollutants. The climate is tropical with annual mean temperatures of 23 ◦ C, with no dry season, high humidity and annual precipitation of 2600 mm at the coastal plain. This is one of the most important industrial complexes of South America, housing 11 petrochemical/chemical industries, 7 fertilizer industries, 1 steel work and 1 cement factory, totaling 206 emission sources of gas pollutants (CETESB, 2002). Highly polluted and non-polluted sites, respectively, Mogi River Valley (MV) and Pil˜oes River Valley (PV) were selected to install the exposure system, based on previous studies that demonstrated differences between the two sites only with regard to the local air contamination. Similar altitudes (around 50 a.s.l.) and meteorological conditions prevail at both sites: they differ with regard to the nature of the local air pollutants (Klumpp et al., 2000; Fiedler and Massambani, 2001; Moraes et al., 2002). MV is in close proximity to the core of the industrial complex, downwind of the industrial complex, and characterized by severe vegetation damage. High contamination of particulate matter, fluorides, sulfur and nitrogen compounds are assumed to predominate at MV (Jaeschke, 2001). Concentrations of 184 g m−3 , 35 g m−3 and 29 g m−3 of total particulate matter, nitrogen oxides and sulfur dioxide, respectively, were reported by the local monitoring station in 2000 (CETESB, 2002). PV site is southwest of the industrial complex and was assumed as the reference area. Low concentration of air pollutants stemming mainly from vehicles characterizes PV. Geographic barriers and location out of reach by the landto-sea breeze determine that the air pollutants emitted by Cubat˜ao industrial complex contaminate only slightly the areas embraced by PV (Jaeschke, 2001). The concentrations of SO2 , fluorides and NOx , the most abundant pollutants at the MV site, were measured at both experimental sites, from March to November 2001,
101
b c
Fluorides (ppm)b
NOx (ppm)c
1 ppm SO2 = 2.67 mg SO2 m−3 . 1 ppm fluoride = 1.58 mg HF m−3 . 1 ppm NO2 = 1.92 mg NO2 m−3 .
by monthly discontinuous samplings with a manual pump (Dr¨agger-Accuro) by a colorimetric approach (Table 1). The accumulated precipitation, the number of unfavorable days for pollutant dispersion and the concentrations of sulfur dioxide (SO2 ), nitrogen dioxide (NO2 ) and particulate matter (MP10) are shown in Table 2 in order to characterize the two periods of experiment (July 2000–June 2001 and December 2000–November 2001) at MV site. It is important to point out that PV is not monitored by the State Agency of Pollution Monitoring (CETESB), since the region is considered non-polluted. 2.2. Plant exposure Saplings of P. guajava L. (about 30 cm high) were obtained from a nursery and planted in plastic pots containing uniform soil. In the field, pots were kept under a shading protection that promoted a 50% reduction of incident sunlight. Thirty-six potted saplings were maintained at both sites during two periods of one year (July 2000–June 2001 and December 2000–November 2001). After 12 months of exposure, undamaged and fully expanded leaves of the fourth node of each sapling were collected for wax chemical and morphological analyses, corresponding to a leaf that expanded after transport to the study sites. The total leaf area used for wax extractions was estimated by scanning leaves using the Scion Image Beta 4.02 program (Scion Corporation, USA). 2.3. Wax morphology Leaves were dried at room temperature. Three inter-venal regions were selected for three saplings of each site to be analyzed. The samples were placed on aluminum-stubs using a double-sided adhesive tape. The specimens were sputter coated with a thin gold-layer and were investigated by scanning electron microscopy (SEM) (ZEISS DSM 940, Germany). To visualize the wax morphology, abaxial and adaxial
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Table 2 Accumulated precipitation (PPT, mm), unfavorable days for pollutant dispersion (UDD) and mean and maximum concentration (g m−3 ) of nitrogen dioxide (NO2 ), sulfur dioxide (SO2 ) and particulate matter (PM10) at Cubat˜ao during periods of exposure PPTa
UDDb
NO2 b Mean
Period I Winter Spring Summer Autumn Total accumulated Annual mean Period II Summer Autumn Winter Spring Total accumulated Annual mean
167.0 692.2 742.0 520.0
31 2 0 13
59.7 49.7 49.0 60.3
2091.2 174.3
46 –
593.0 53.9
742.0 431.1 259.5 569.4
0 4 30 3
49.0 60.3 77.0 57.7
2002.0 166.8
37 –
665.0 59.5
SO2 b Maximum 110.0 94.0 88.0 113.0
Mean 43.3 23.3 20.7 27.7
PM10b Maximum 114.0 49.0 59.0 77.0
328.0 27.3 88.0 113.0 176.0 101.0
20.7 27.7 35.3 24.0
Mean 183.3 162.3 212.9 197.9
Maximum 347.0 320.0 368.0 407.0
2279.6 190.0 59.0 77.0 112.0 96.0
323.0 26.9
212.9 197.9 244.1 231.1
368.0 407.0 587.0 306.0
2658.2 221.5
Period I: July 2000–June 2001; Period II: December 2000–November 2001. a Data from Empresa Metropolitana de Aguas ´ e Energia (EMAE). b Data from Companhia de Tecnologia e Saneamento Ambiental (CETESB).
leaf surfaces were observed in secondary electron mode, at an acceleration voltage of 10 keV and magnification of 1000×. The wax morphology of the abaxial and adaxial leaf surfaces was investigated before and after the wax chloroform extraction, described in the next section. 2.4. Wax analysis Foliar waxes of all saplings were extracted with chloroform by three consecutive immersions of 10 s each. The extracts were filtered through chloroform-washed filter paper and the solvent was evaporated under reduced pressure. The extracted waxes were weighted and the alkane fraction was separated by column chromatography (CC) using aluminum oxide and n-hexane. Alkanes were analyzed by gas chromatography and mass spectroscopy, using a capillary column DB-5HT HP (30 m, 0.32 mm), helium at a flow rate of 1 mL min−1 and electronic impact method at 70 eV. The temperatures of the injector and the detector were 300 ◦ C. The temperature of the column ranged between 140 ◦ C (1 min) and 300 ◦ C at 10 ◦ C min−1 . The temperatures of the MS source and quadruple were, respectively, 250 ◦ C and 100 ◦ C. The identification of the individual alkanes was achieved by the corresponding mass spectra and comparison of retention times with those of n-alkane standards. 2.5. Statistical analysis The percentage data were transformed into the corresponding square roots for each value. The raised data were statistically treated using ANOVA. The procedure of pairwise comparison (Bonferroni’s test) was used when ANOVA revealed significant differences between sites and year periods.
3. Results and discussion The morphology of abaxial and adaxial leaf surfaces of P. guajava after 12 months of exposure to air pollutants emitted by the industrial complex of Cubat˜ao is shown in Fig. 1. The morphology of leaf surfaces before and after chloroform extraction of total wax showed no differences, suggesting that the leaf epidermal cells in P. guajava are covered by a thin wax layer. Total wax contents (TWC) were not significantly different in leaves of saplings exposed at both sites (Table 3). On the other hand, higher quantities of wax are noted for saplings in both sites comparing the periods December 2000–November 2001 and July 2000–June 2001. These results suggest a stronger climatic influence on wax production than a pollution effect. Faini et al. (1999) studying seasonal changes in chemical composition of epicuticular waxes of leaves of Baccharis linearis observed higher production of epicuticular waxes in summer, when drought and solar radiation are highest. Higher production of epicuticular waxes in summer may be advantageous inasmuch as the waxes may act as a physical barrier to prevent water permeation and dehydration of the leaves, in addition to filtering and reflecting solar radiation. According to Riederer and Schreiber (2001), the cuticle is the major barrier against uncontrolled water loss from leaves, fruits and other parts of higher plants. It is known that the water permeability of the plant cuticle is determined by wax composition and ultrastructure. Studies involving extraction of total foliar waxes showed an increase of the cuticle permeability to sulfur dioxide, hydrogen fluoride, carbon dioxide and herbicides (Poborski, 1988; Santier and Chamel, 1998). Plants under water stress have been found to have an increase of the waxes total amount (Poborski, 1988; Bondada et al., 1996). In addition, leaves of
C.M. Furlan et al. / Environmental and Experimental Botany 58 (2006) 100–105
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Fig. 1. Scanning electron microscopy of leaves of Psidium guajava exposed for 12 months at two areas: (A) abaxial and (B) adaxial leaf surfaces of saplings exposed at control site (PV), respectively; (C) abaxial and (D) adaxial leaf surfaces of saplings exposed at polluted site (MV), respectively; (E) adaxial leaf surface after wax extraction. Bars: 10 m. Note abundant deposits of particulate pollutants in (D).
heavily polluted areas exhibit enhanced water loss simultaneously with changes in the wax composition and ultrastructural alterations (Cape and Percy, 1998; Bytnerowicz et al., 1998; Viskari et al., 2000). It was noted that not only the amounts of wax are important to protect the leaf tissues against water
loss, but also their chemical composition, in especially long chain aldehydes, free primary alcohols and other non-polar substances (Poborski, 1988; Bondada et al., 1996). The n-alkane wax homologues of P. guajava after 12 months of exposure in both areas were predominantly C25 ,
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Table 3 Relative abundance (%) of n-alkanes and total wax content (TWC) of leaves of Psidium guajava saplings (N = 36) exposed for two periods of 12 months at polluted (MV) and non-polluted (PV) sites Chain length
July 2000– June 2001
December 2000– November 2001
PV
MV
PV
MV
C19 C20 C21 C22 C23 C24 C25 C26 C27 C28 C29 C30 C31 C19 –C23 C24 –C29 Codd Ceven Even/odd C19 –C23 /C24 –C29
0.16a 1.54a 0.37a 3.41a 4.48a 9.70a 20.56a 20.06a 22.96a 5.53a 8.98a 0.97a 1.28a 9.96a 90.04a 58.79a 41.21a 0.70a 0.11a
0.28a 2.64a 1.25b 4.77b 7.35b 11.38a 18.38a 16.68b 20.98b 7.18b 7.16a 0.98b 0.99a 16.28b 83.72b 56.38b 43.62a 0.77b 0.19b
0.00a 0.81a 0.00a 5.44a 3.52a 14.19a 16.29a 23.59a 20.99a 6.43a 7.30a 1.34a 0.10a 9.77a 90.23a 48.20a 51.80a 1.07a 0.11a
1.09a 0.71a 0.50a 8.57a 6.33b 13.44a 17.34a 20.14b 18.44a 7.30a 4.65a 1.38a 0.07a 17.22a 82.78a 48.44a 51.56a 1.06a 0.21a
TWC (g cm−2 )
34.6b
40.1b
73.8a
88.6a
Values followed by different letters (a and b) correspond to different means (P ≤ 0.05) between PV and MV at the same exposure period. For TWC, values followed by different letters (a and b) correspond, for the same site, to different means (P ≤ 0.05) between July 2000–June 2001 and December 2000–November 2001.
C26 and C27 (Table 3). However, an increase of the relative amounts of homologues with shorter carbon chains (C19 –C23 ) and a decrease of the relative amounts of homologues with longer carbon chains (C24 –C31 ) were noted in the foliar waxes of saplings under pollutants influence (Table 3) exposed in the period starting in the winter (July). Although not significant, the values observed for the second period of exposure showed a similar trend toward higher amounts of homologues with shorter chains. Even/odd and short/long carbon chains ratios of leaf waxes increased in MV in the first period of exposure (July 2000–June 2001; Table 1). The climatic conditions of the first months of exposure seem to be important to establishing the intensity of plant responses to air pollution. In the experiment started in the winter, the plants grew over the first six months under drier atmosphere and more unfavorable conditions for pollutant dispersion, reaching the rainy summer and autumn periods only in the last six months. On the other hand, the second period of exposure (December 2000–November 2001) corresponds to summer and autumn for the first six months and to winter and spring for the final months. The results suggest that harsher growth conditions (low moisture and more pollutants) in the first months of exposure are more effective to alter wax composition than the milder conditions prevalent in the wetter seasons.
Turunen et al. (1997) studied the response of epicuticular wax of Pinus sylvestris needles to dry and wet deposited sulfur and heavy metals found that sulfur deposition plays a more important role in needle surface deterioration than heavy metals. Sulfur dioxide and acid rain have been found to be more deleterious to needles surfaces than ozone (Cape et al., 1995). Significant differences in n-alkanes composition occurred only during the first exposure period, when atmospheric sulfur dioxide concentration was highest (Table 3). Kerfourn and Garrec (1992) studied Hedera helix and Picea abies exposed to acid and ozone found higher amounts of short chain and lower amounts of long chain alkanes in P. abies, as well as increased ratios of even to odd chains for H. helix leaves exposed to acid rain. Barnes et al. (1994) studied Nicotiana tabacum exposed to enhanced UV-B radiation also found shifts of n-alkane composition toward shorter chain lengths. Turunen et al. (1997) found effects on wax chemical composition of P. sylvestris due to the proximity of a smelter industry. Significant site influences were found especially for secondary alcohols, the major constituent of P. sylvestris waxes. The results observed in saplings of P. guajava exposed to air pollutants are similar to those found by Kerfourn and Garrec (1992). The alteration might be caused by the presence of higher concentrations of SO2 and NOx in Cubat˜ao, which contribute to rain acidification (CETESB, 2002). The alterations on alkane composition are particularly interesting because of their relationship with foliar penetration by chemicals, particularly taking into account that they belong to the most hydrophobic class of plant waxes. Long chain alkanes are known to be more resistant to the penetration of polar solutions than shorter homologues, in addition to playing an important role as plant protectors against adverse environmental conditions, such as water stress (Kerfourn and Garrec, 1992; Bianchi, 1995; Bondada et al., 1996). Oliveira et al. (2003) verified that the chemistry of the wax constituents is a factor more important to determine the degree of resistance to evaporation than the thickness of the waxy deposits. The authors verified that n-alkanes and alcoholic triterpenes were more efficient barriers to water loss than hentriacontan16-one (a ketone) and ursolic acid (an acid triterpene). Oliveira and Salatino (2000) investigated the major constituents of foliar waxes of species from caatinga (semi-arid ecosystem in northeast Brazil) and cerrado (savanna ecosystem in central and southeast Brazil). Although n-alkanes and triterpenoids are important constituents of waxes of both habitats, the former substances were found to be more common and abundant in plants from the caatinga. The present results are the first report about effects of air pollutants on n-alkane distributions in leaf waxes of a tropical tree species. A question may be raised at this point whether the observed induced distribution may have reflections on alterations of the efficiency of water retention. Studies combining these approaches are important to integrate the influence of pollution stress and other environmental factors.
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Acknowledgements The authors thank FAPESP (Proc. No. 99/06971-9) and CNPq for provision of funds, and Instituto Florestal (N´ucleo Pil˜oes) and Cesari Ltda for allowing the use of the areas for exposure of the saplings.
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