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Critical analysis of the potential of Ipomoea nil ‘Scarlet O’Hara’ for ozonebiomonitoring in the sub-tropics

Maur�ıcio Lamano Ferreira,a J�essica Bordotti Nobre Esposito,b Silvia Ribeiro de Souzab and Marisa Domingos*b

Received 13th January 2012, Accepted 27th April 2012

DOI: 10.1039/c2em30026e

This study aimed to analyze critically the potential of Ipomoea nil ‘Scarlet O’Hara’ for O3 biomonitoring

in the sub-tropics. Four field experiments (one in each season of 2006) were carried out in a location of

the city of Sao Paulo mainly polluted by O3. Each experiment started with 50 plants, and lasted 28 days.

Sub-lots of five plants were taken at intervals between three or four days long. Groups of four plants

were also exposed in closed chambers to filtered air or to 40, 50 or 80 ppb of O3 for three consecutive

hours a day for six days. The percentage of leaf injury (interveinal chloroses and necroses), the

concentrations of ascorbic acid (AA) and the activity of superoxide dismutase (SOD) and peroxidases

(POD) were determined in the 5th, 6th and 7th oldest leaves on the main stem of the plants taken in all

experiments. Visible injury occurred in the plants from all experiments. Seasonality in the antioxidant

responses observed in plants grown under field conditions was associated with meteorological variables

and ozone concentrations five days before leaf analyses. The highest levels of antioxidants occurred

during the spring. The percentage of leaf injury was explained (R2 ¼ 0.97, p < 0.01) by the reduction in

the levels of AA and activity of POD five days before the leaf analyses and by the reduction in the levels

of particulate matter, and enhancement of temperature and global radiation 10 days before this same

day. Although I. nil may be employed for qualitative O3 biomonitoring, its efficiency for quantitative

biomonitoring in the sub-tropics may be compromised, depending on how intense the oxidative power

of the environment is.

Introduction

Ozone (O3), upon entering the leaves via stomata, enhances the

formation of reactive oxygen species (ROS) in plant tissues.

These react indiscriminately with biomolecules, causing the

appearance of interveinal leaf chlorosis, necrosis and punctate

lesions in sensitive plant species, among many other possible

injuries.1–6 The analysis of these visible symptoms has been

aUniversidade Nove de Julho, Faculdade de Biologia, Av. Adolfo Pinto 109,01156-050 Sao Paulo, SP, BrazilbInstituto de Botanica, Caixa Postal 68041, 04045-972 Sao Paulo, SP,Brazil. E-mail: mmingos@superig.com.br

Environmental impact

The present study revealed that Ipomoea nil ‘Scarlet O’Hara’, a nativ

for qualitative O3 biomonitoring in the sub-tropics. It showed typica

moderate O3 levels observed during the field experiments performed

the percentage of visible leaf injury could not be directly associated

association occurred among environmental changes, antioxidant

efficiency for quantitative O3 biomonitoring may be compromised in

of the environment is.

This journal is ª The Royal Society of Chemistry 2012

successfully employed for O3 biomonitoring in the northern

hemisphere, mainly due to its feasibility of application and high

reliability of responses.6–11

This alternate method for O3 monitoring may also be an

important tool in the sub-tropics, where many megacities, such

as Sao Paulo, exist, and the instrumental O3 monitoring is not as

widespread as desirable.12,13 Nicotiana tabacum ‘Bel W3’ is the

most common sensitive plant for O3 biomonitoring.7,8,14

However, it is questionable whether the use of such a supersen-

sitive bioindicator is appropriate for estimating the O3 levels in

the atmosphere because of the inherent multicollinearity of

effects caused by other environmental variables together with

ozone.15 Sant’Anna et al.,16 Esposito et al.,17 Dias et al.,18 and

e climbing species of the tropical Americas, might be employed

l interveinal chloroses and necroses on the older leaves under the

in a location of Sao Paulo (SE Brazil). However, the increase in

with increased levels of O3. A hierarchical and time-dependent

responses and the occurrence of visible injury. Therefore, its

the sub-tropics, depending on how intense the oxidative power

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Alves et al.19 showed that short-term variations in the meteoro-

logical conditions generally observed in theMetropolitan Region

of Sao Paulo (MRSP), SE Brazil, may modulate some leaf

physiologic, metabolic and structural responses in plants of this

cultivar, which may interfere with the intensity of leaf necroses.

Dias et al.18 and Alves et al.19 added that the redox state of

tobacco Bel-W3 decreases a few days after their placement in

a subtropical environment contaminated by low doses of O3,

then microscopic and macroscopic damages emerge due to

oxidative stress. Sant’Anna et al.16 also verified that the disrup-

tion of the ascorbate–glutathione cycle in tobacco plants exposed

to higher doses of O3, in a fumigation chamber, appears to be

followed by increasing physiological damage, affecting the

pollutant uptake in such a way that a weak relationship between

leaf necroses and O3 would be one probable practical result.

Therefore, a less sensitive bioindicator species might be appro-

priate in this case. In theory, a native species to the sub-tropics

would be more adequate for this purpose because it would better

tolerate the short-term variations in the meteorological

conditions.

The cultivar ‘Scarlet O’Hara’ of Ipomoea nil, a native climbing

species of the tropical Americas, is one possible alternative bio-

indicator because it developed typical visible injuries (interveinal

chloroses and necroses) after experimental exposure to 196 ppb

of O3 for one hour or to 76 ppb for eight hours.20 Nevertheless,

Dafr�e-Martinelli et al.21 concluded that the bioindicator effi-

ciency of this cultivar, exposed in locations of Sao Paulo

contaminated by low levels of O3, may be restricted by the

increased tolerance to oxidative stress, mainly conferred by

ascorbic acid and glutathione, although this enhanced tolerance

did not prevent the appearance of typical visible injury on the

leaves. But, these symptoms were only interveinal chloroses,

whose severity is difficult to estimate from a practical point of

view. Therefore, the following critical questions were raised from

this last study: Would the increase in the levels of antioxidants

prevent the occurrence of a more specific leaf visible injury in

plants of I. nil ‘Scarlet O’Hara’ growing under the moderate to

high levels of O3 generally observed in the sub-tropics? Would

this antioxidant modulation of leaf visible injury be seasonally

marked? The present study was directed to answer these ques-

tions, aiming to analyze critically the potential of Ipomoea nil

‘Scarlet O’Hara’ for O3 biomonitoring in the sub-tropics. During

the four seasons of the year we assessed both the intensity of

leaf injury and some indicators of the antioxidant system

throughout the development of plants exposed to fluctuating

environmental conditions in a location in the city of Sao Paulo

mainly polluted by O3. We also checked, under fumigation

conditions, whether the leaf injury and the antioxidant responses

observed in the plants exposed in the field might be attributed at

least in part to O3.

Materials and methods

Plant cultivation

The cultivation of the plants used in all field and fumigation

experiments followed the same procedures. They were obtained

from seeds commercially acquired (from CN Seeds LTD,

www.cnseeds.co.uk), which were germinated in a mixture of

1960 | J. Environ. Monit., 2012, 14, 1959–1967

a commercial substrate mainly composed of bark of Pinus

(Plantimax-Eucatex) and fine vermiculite in the ratio of 3 : 1,

respectively. The resulting seedlings were transplanted to plastic

vases with the same substratum mixture and were maintained

inside a greenhouse under filtered air, provided by activated

charcoal and filters for particulate material, until the beginning

of each experiment. The plants were continuously watered by

means of strings inserted into the bottom of the vases at one end

and immersed in water reservoirs at the other. They were also

supplemented weekly during the cultivation with a fertilizer

solution prepared according to Epstein.22

Field experiments

The field experiments were conducted at Ibirapuera Park,

a public area located in the city of Sao Paulo, SE Brazil

(23�340550 0 N latitude, 46�390250 0 W longitude, 750 meters over

sea level). The park is located between avenues with heavy

vehicular traffic and historically affected by high and rising levels

of O3, especially in the seasons of spring and summer, and lower

levels of pollutants emitted by the vehicle fleet.23 The climate is

subtropical, with an average annual temperature of 18.3 �C, withmild winters and summers with high temperatures. The relative

humidity is usually above 60%. The average annual rainfall is

1368 mm, concentrated mainly in the summer.24

Four field experiments were carried out, one in each season of

2006. The summer, autumn, winter and spring experiments were

performed in February/March, May/June, August/September

and November/December, respectively. During all the experi-

mental periods, the plants also had adequate irrigation guaran-

teed by capillarity through nylon strings, received periodic

fertilization with an aqueous nutrient solution prepared

according to Epstein,22 and were maintained in shaded appara-

tuses (50% reduction of sunlight).

Each field experiment started with 50 plants with at least seven

fully expanded leaves, including the cotyledonal ones, and lasted

28 days. During this period of 28 days, sub-lots of five plants

were taken in the morning (around 10 a.m.), at intervals between

three or four days long (across a total of nine sampling days). The

percentage of leaf area covered by typical O3-induced injury, the

concentrations of ascorbic acid (AA) and the activities of

superoxide dismutase (SOD) and peroxidases (POD) were

determined in the 5th, 6th and 7th oldest leaves on the main stem of

the plants taken in each sampling day. An additional group of

five plants was taken for the determination of the antioxidant

profile at time zero of each field experiment.

Fumigation experiments

The fumigation experiments were all performed during 2006 in

closed chambers with dimensions of 85 � 94 � 85 cm (W � D �H) that were composed of a stainless steel structure covered by

a film of Teflon�. They were located inside a laboratory and

received artificial light supplied by metallic vapor and fluorescent

lamps. The chamber design and functioning were described in

detail by Souza and Pagliuso.25

Groups of four plants were concurrently exposed to filtered air

by means of silica gel, activated charcoal and Purafil blend

(control treatment) or filtered air enriched with 40, 50 or 80 ppb

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of O3 (O3 treatment) for three consecutive hours a day (between

9 am and 12 pm) for six days. The Purafil blend eliminated

almost 90% of nitrogen oxide, decreasing the concomitant

production of nitrogen oxidants and O3 and ensuring the high

quality of the control treatment. Input O3 levels were continu-

ously measured with an Ecotech� 9810Bmonitor. The chambers

received equivalent fluxes of air during the fumigation. Two

replications of each O3 treatment experiment were performed.

The percentage of leaf area covered by typical O3-induced injury

and the concentrations of AA and the activities of SOD and POD

were determined in the 5th, 6th and 7th oldest leaves of the main

stem at the end of each experiment.

In particular, one replication of the fumigation experiment

with 80 ppb of O3 occurred at the end of autumn and the other in

the middle of winter.

Analysis of leaf visible injury and antioxidant indicators

The percentage of the injured leaf area in each field or fumigation

experiment was estimated in classes of 5% following the proce-

dure proposed for Nicotiana tabacum ‘‘Bel-W3’’ by the Verein

Deutscher Ingenieure (VDI).26

Ascorbic acid was measured in fresh leaves, homogenized with

EDTA-Na2 (0.07%) and oxalic acid (0.5%), following the

method described by Keller & Schwager.27 The extract was

centrifuged at 40 000g for 30 min at 2 �C. An aliquot of the

supernatant was added to DCPiP (0.02%), measuring the

absorbance at 520 nm with a spectrophotometer. The absor-

bance was measured again after the addition of ascorbic acid

(1%). The concentration of ascorbic acid content was estimated

based on the difference of both absorbance measurements.

Superoxide dismutase activity was measured in 0.1 g of fresh

leaves that was homogenized with 12 mL of potassium phos-

phate buffer (50 mM, pH 7.5), 1 mM EDTA-Na2, 50 mM NaCl

and 1 mM ascorbic acid in the presence of 0.4 g of 2% PVPP. This

mixture was centrifuged at 22 000g for 25 min at 2 �C. Theactivity of SODwas assayed by measuring the SOD inhibition by

NBT photochemical reduction. This method was adapted from

Osswald et al.28

The peroxidase activity was determined in a reaction mixture

of plant extracts using 0.1 M potassium phosphate buffer (pH

5.5) and phenylenediamine (1%), to which an aliquot of H2O2

(0.3%) was added. Unspecific POD activity was measured with

a spectrophotometer following the increase in absorbance (DA)

at 485 nm due to the formation of an H2O2–POD complex at two

different times in the linear reaction curve.29,30

Statistical analyses

Differences in antioxidant responses among leaves for each

sampling day of each seasonal field experiment, as well as

differences between the seasons, were determined by means of

the Kruskal–Wallis One Way Analysis of Variance. In all cases,

a post-hocmultiple comparison test (Dunn test) was applied if the

analysis of variance indicated significant differences. The anti-

oxidant responses in plants that were subjected to control and

ozone treatments in each fumigation experiment were compared

by means of the T-test. Similar comparisons were performed

between the autumn and winter experiments with 80 ppb of O3.

This journal is ª The Royal Society of Chemistry 2012

A canonical correspondence analysis (CCA) was performed to

summarize the overall variability of the data from the field

experiments, after their transformation by log 10, and qualita-

tively evaluate the relationship between the biological and envi-

ronmental factors. Similarly, a principal component analysis

(PCA) was applied on data from the fumigation experiments to

summarize the overall variability and qualitatively evaluate the

relationship between the leaf injury and antioxidant indicators

analysed.

Multiple linear regression analyses were performed in order to

ascertain if the antioxidant responses in plants growing at Ibir-

apuera Park might be predicted by oscillations in meteorological

variables (air temperature, relative humidity, global radiation

and wind speed) and/or the pollutant concentrations (PM10, NOx

and O3), continuously measured by a monitoring station of the

State Company of Air Pollution Control (CETESB), installed

next to the apparatus for plant exposure. Matrices of Pearson’s

correlation between the biological variables measured at each

day of analysis in each season and the average environmental

conditions zero to ten days prior to the leaf analyses were

preliminarily constructed. The highest and significant correlation

coefficients were found between the biological variables and the

environmental conditions five days prior to the leaf analyses

(data not shown). Therefore, only this set of data was sequen-

tially used for performing the multivariate analyses to identify

the environmental variables that significantly explained the

variations in the biological responses. The data of all the seasonal

campaigns were jointly analyzed through the stepwise backward

method, adopting the antioxidant responses as dependent vari-

ables (after appropriate transformation when necessary) and the

environmental conditions (five days before the antioxidant

analyses) as independent variables. The adjustment procedure

for each set of data started from the saturated model (with all the

variables present) and consisted of removing the variable with

the smallest participation to explaining the variations in anti-

oxidant responses. New adjustments were successively made. At

each step, the adjustment and the significance of the variables

were evaluated, and only those that contributed significantly to

explaining the remaining variation were retained.

Finally, the relationship between the percentage of leaf injury

and doses of exposure to O3, expressed as accumulated doses

over 20 ppb (AOT20), was tested by performing a linear

regression analysis.

The statistical, ordination and multiple regression analyses

were performed in accordance with Kutner et al.31 and Manly,32

employing the software SigmaStat for Windows 3.5 (variance

analyses and the linear regressions) or Software PC-ORD 5.15

for Windows (CCA and PCA).

Results

Environmental conditions

(a) Field experiments. The environmental conditions during

the field experiments varied markedly among seasons. The

average values of temperature at Ibirapuera Park were higher

during the spring and summer experiments. The relative

humidity was higher during the summer and lower during the

winter. The lowest values of global radiation were observed

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during the autumn and the highest values were observed during

the summer and winter field experiments (Table 1).

During 2006, the region of the park presented the highest

average concentration of O3 in the summer, with an average of

32.4 ppb. On the other hand, the winter was the season with

lowest values of O3, with a maximum and minimum of 28.8 ppb

and 11.1 ppb, respectively, and an average of 20.2 ppb. Although

the highest average level of O3 at Ibirapuera Park was observed

during the summer, the highest AOT40 was estimated during the

winter experiment. The average concentrations of PM10 and NO2

were higher during the autumn and winter than during the other

seasonal campaigns (Table 1).

(b) Fumigation experiments. Although the photosynthetic

photon flux density did not vary during the fumigation experi-

ments (84.2 � 8.7 mmol m�2 s�1), average temperatures inside the

laboratory were similar during the short periods of both repli-

cations, but the difference between the maximum and minimum

daily values was smaller during the first replication performed in

the autumn (26.4–7.9 �C) than during the second, which was

conducted in the winter time (29.4–7.0 �C).

Leaf injury

Visible injuries were always interveinal, occurred on the adaxial

surface of the older leaves and were observed during all field and

fumigation experiments (Fig. 1). The leaf injury was character-

ized by chlorotic spots (Fig. 1A andD), necrotic spots covered by

spread and chlorosis irregularly shaped (Fig. 1B and E) or by

spread and irregular necroses (Fig. 1C and F).

The leaf injury always appeared during the second week of

exposure at Ibirapuera Park. The average percentage of injured

leaf area was higher and progressive during the summer experi-

ment, reaching values above 30% at the end of exposure. In the

autumn, a low average percentage of leaf injury was observed

(<5%). During the winter and spring experiments, around 10% of

leaf area exhibited chlorosis and/or necrosis (Fig. 2).

Fig. 1 Sixth leaf of different plants of I. nil ‘Scarlet O’Hara’, indicating

the occurrence of visible leaf injury during the field (A, B, and C) and

fumigation (D, E, and F) experiments. (A and D). Leaves with interveinal

chlorotic spots. (B, and E). Leaves with necrotic spots surrounded by

spread interveinal chlorosis. (C and F). Leaves predominantly affected by

interveinal necrosis.

Antioxidant responses of plants

(a) Field experiments. In the majority of the cases, no signifi-

cant differences in antioxidant responses were found among the

5th, 6th, and 7th oldest leaves analyzed on each sampling day

during each field experiment. Therefore, the antioxidant

responses are presented as the average per plant. Only data from

Table 1 Average values of environmental variables at Ibirapuera Park during the four field experiments carried out in 2006. Minimum and maximumdaily average values are between parentheses. T: air temperature; RH: relative humidity; GR: global radiation; WS: wind speed; O3: ozone; AOT40(accumulated doses of O3 over 40 ppb); PM10: particulate matter # 10 mm. Data from the monitoring station belonged to the State Agency of AirPollution Control of Sao Paulo – CETESB (http://www.cetesb.sp.gov.br/ar/qualidade-do-ar/31-publicacoes-e-relatorios)

Environmental variables Summer Autumn Winter Spring

T (�C) 23.0 (20.3–25.0) 18.3 (16.0–20.0) 19.5 (13.9–24.0) 21.6 (19.4–25.4)RH (%) 82 (70–98) 81 (77–88) 74 (56–90) 82 (64–97)GR (W m�2) 381 (145–577) 176 (104–220) 352 (56–488) 181 (77–328)WS (m s�1) 1.2 (0.7–2.5) 1.0 (0.4–2.1) 1.5 (0.5–2.9) 1.4 (0.8–2.6)O3 (ppb) 32.4 (0.6–55.3) 27.5 (11.2–55.3) 20.2 (11.1–28.0) 26.6 (10.3–48.8)AOT40 (ppb h) 1170 173 1412 721PM10 (mg m�3) 32.2 (13.5–55.4) 51.0 (0.5–91.1) 47.2 (10.8–99.8) 27.2 (10.2–63.1)NO2 (mg m�3) 41.4 (17.3–77.1) — 63.0 (31.2–115.0) 48.5 (27.3–68.5)

1962 | J. Environ. Monit., 2012, 14, 1959–1967 This journal is ª The Royal Society of Chemistry 2012

Fig. 2 Average in percentage of injured leaf area (% INJ) of the 5th, 6th

and 7th oldest leaves of the main stem, in plants of I. nil ‘Scarlet O’Hara’

exposed at Ibirapuera Park over the 28 days of each field experiment.

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measurements performed every seven days during each field

experiment are presented in Table 2. This table also contains the

average values per season.

The leaf concentrations of AA and the enzymatic activities

in the plants of I. nil ‘Scarlet O’Hara’ generally oscillated

throughout the field experiments. The contents of AA, during the

summer, reached the maximum during the second week of

exposure and then gradually decreased. The leaf AA concen-

tration remained almost constant during plant growth in the

autumn experiment. Significantly higher concentrations of AA

were measured on the last day of sampling in the winter experi-

ment. In the spring, leaf AA varied throughout all 28 days of the

experiment, reaching maximum values after 14 days. On average,

the highest leaf levels of AA were observed during the spring

experiment. Additionally, a markedly seasonal profile in the

concentrations of AA was found in plants growing at Ibirapuera

Park (summer < winter ¼ autumn < spring).

The leaf activity of SOD was significantly higher between 7

and 21 days of plant exposure under field conditions, during the

summer and autumn experiments, and it gradually increased

until the end of both the winter and spring field experiments. On

average, significantly higher values of SOD activity were

obtained in the plants grown during the 28 days of spring.

The activity of POD did not differ significantly in plants

sampled during either the autumn or winter experiments.

Table 2 Average values of the concentrations of ascorbic acid (AA) and the aI. nil ‘Scarlet O’Hara’ grown at Ibirapuera Park, in the beginning (0) and afte2006. Distinct lower-case letters indicate significant differences among daysindicate significant differences among seasons for each antioxidant indicator

Antioxidant indicators Seasons

Days of exposure

0 7

AA (mg g�1 DW) Summer (C) 2.1 d 11Autumn (B) 6.9 a 5.8Winter (B) 5.9 b 6.8Spring (A) 9.1 a 8.4

SOD (Units g�1 DW) Summer (B) 134 b 48Autumn (B) 298 c 41Winter (B) 287 b 55Spring (A) 282 d 41

POD (dE min�1 g�1 DW) Summer — —Autumn (C) 957 a 12Winter (B) 987 a 15Spring (A) 1120 b 22

This journal is ª The Royal Society of Chemistry 2012

However, POD showed a clear oscillatory profile during the

spring experiment, showing higher activity after 7, 14 and 28 days

of exposure (Table 2). A markedly seasonal profile in the

activity of POD was also found in plants grown in the field

(autumn < winter < spring).

(b) Fumigation experiments. The exposure of plants to 80 ppb

of ozone for 6 days (three hours per day) caused a significant

reduction in the concentrations of AA in both fumigation

experiments. The activity of SOD was not significantly altered

after ozone exposure. A significant decrease in the activity of

PODwas observed only at the end of the autumn experiment. No

seasonal effect was observed in the levels of AA in plants exposed

to filtered air or ozone treatment. However, the activity of

SOD and POD was seasonally affected. The SOD activity was

significantly higher during the autumn than during the winter in

plants from both fumigation treatments. POD activity was

seasonally marked only in the plants from the ozone treatment

(winter > autumn) (Fig. 3).

Relationships among environmental variations, leaf injury and

antioxidant in the plants

(a) Field experiments. The canonical correspondence analysis

(CCA) was done with all the data obtained during the four field

experiments (Fig. 4). It extracted 18.3% of the total variability of

the data in the first two axes (laxis 1 ¼ 0.092; laxis 2 ¼ 0.023). The

high Pearson correlation between the biological matrix and the

environmental matrix for axes 1 (r ¼ 0.57) and 2 (r ¼ 0.50)

indicated that both matrices are significantly related, which was

confirmed by the Monte Carlo permutation test (p ¼ 0.03 and

p ¼ 0.02, for axes 1 and 2 respectively). The global radiation was

the major variable in the ordination of axis 1 (r ¼ 0.81).

Temperature and ozone were the main variables in the ordina-

tion of axis 2 (r ¼ 0.83 and r ¼ 0.76, respectively).

All sample units from the experiments performed during the

summer and winter were placed on the positive side of axis 1 and

were associated with higher values of solar global radiation. In

ctivity of superoxide dismutase (SOD) and peroxidase (POD) in plants ofr 7, 14, 21 and 28 days of exposure in each field experiment carried out inof exposure in each season (Dunn test; p < 0.05). Distinct capital letters(Dunn test; p < 0.05)

Average14 21 28

.2 a 7.1 b 6.3 c 3.0 c 5.9b 5.8 b 7.8 a 6.9 a 6.6b 5.8 b — 9.1 a 6.9a 11.3 a 8.3 a 8.5 a 9.1

0 a — 600 a 270 b 3714 bc 464 bc 926 a 200 d 4601 b 150 b — 784 a 4432 c 640 b 918 a 863 a 623

— — — —14 a 905a 306 a 938 a 86477 a 1747a — 1638 a 148739 a 2434a 1420 b 1627 a 1768

J. Environ. Monit., 2012, 14, 1959–1967 | 1963

Fig. 3 Box-plots of the level of antioxidant indicators measured in

plants of I. nil ‘Scarlet O’Hara’ after exposure to filtered air (control) or

to ozone (80 ppb) in the autumn and winter of 2006. (A). Ascorbic acid

(AA). (B). Superoxide dismutase (SOD). (C). Peroxidase (POD). Distinct

letters indicate significant differences among medians between seasons

within the same treatment (T-test; p < 0.05). The median in the box

signaled with an asterisk (*) is significantly higher than the median in the

box of the other fumigation treatment, within the same season (p < 0.05).

Fig. 4 CCA (canonical correspondence analysis) biplot of the environ-

mental variables (represented by the vectors) and of the biological

responses (represented by asterisks) measured in plants of I. nil ‘Scarlet

O’Hara’ exposed at Ibirapuera Park during the seasonal field experi-

ments. T: air temperature; GR: global radiation; MP10: particulate

matter # 10 mm; O3: ozone; AA: ascorbic acid; SOD: superoxide dis-

mutase; POD: peroxidase; INJ: percentage of leaf injury.

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general, the SOD activity and the occurrence of leaf injury were

in connection with these sample units. The sampling units from

the autumn and spring experiments appeared on the negative side

of axis 1, in a stronger association with high values of MP10, the

highest AA concentration and POD activity, and a lower activity

of SOD and intensity of leaf injury. Therefore, axis 1 of the CCA

separated the climatic seasonality and axis 2 the air pollution

1964 | J. Environ. Monit., 2012, 14, 1959–1967

seasonality. In summary, the CCA also stated that the intensity

of leaf injury was associated with atmospheric ozone concen-

trations during the period of the study, but the relationships

between these variables were mediated by the antioxidative

responses of plants exposed in the field.

The stepwise multivariate analysis, performed with the same

data used in the CCA, showed that variations in the three anti-

oxidants in plants exposed during the year of field experiments

were significantly explained (p < 0.01) by a linear combination of

meteorological variables and pollutants five days before the

sampling days. The most explicative model was proposed for

the activity of POD (r2 ¼ 0.57) and the least explicative for the

activity of SOD (r2 ¼ 0.20). The concentrations of AA increased

following the reduction of relative humidity and global radiation

(indicated by a negative relationship) and the enhancement of the

level of ozone in the atmosphere (positive relationship) five days

before the leaf analyses. The activity of SOD increased in

response to decreased solar radiation (negative relationship) and

increased wind speed and concentrations of particulate material

(positive relationship). The activity of POD was higher following

increases in the average temperature, relative humidity, wind

speed and ozone (positive relationship) (Table 3).

The average percentage of leaf injury for all plants sampled

from each sampling day (data from all field experiments treated

jointly) was preponderantly explained (R2 ¼ 0.97, p < 0.01) by

the reduction in the average levels of AA and activity of POD in

plants sampled five days prior to the leaf analyses and by the

reduction in the daily levels of particulate matter (MP10), and

the enhancement of average of temperature (T) and global

This journal is ª The Royal Society of Chemistry 2012

Table 3 Predictive linear models between antioxidant responses or leaf injury in plants of I. nil ‘Scarlet O’Hara’ and environmental factors five daysprior to leaf sampling at Ibirapuera Park in 2006 (data of all experimental experiments included) and linear regression between leaf injury and accu-mulated doses of O3 over 20 ppb under fumigation conditionsa

Predictive equations R2 (F test; p < 0.01)

Field experimentsAA ¼ [C] � RH � GR + O3 0.33log10SOD ¼ [C] � GR + WS + MP10 0.20SqrtPOD ¼ [C] + T + RH + WS + O3 0.57INJ ¼ [C] � AA5d.b. � POD5d.b. � MP10d.b. + T10d.b. + GR10d.b. 0.97Fumigation experimentsINJ ¼ �3.16 + 0.03 � AOT20 0.84

a AA ¼ Ascorbic acid; SOD ¼ superoxide dismutase; POD ¼ peroxidase; C ¼ constant; INJ: percentage of leaf injury. RH, GR, WS, T and MP10 ¼daily average of relative humidity, global radiation, wind speed, temperature and particulate material 10 mm respectively; O3 ¼ daily average of hourlymaximum concentrations of ozone; AOT20¼ accumulated doses of O3 over 20 ppb. 5d.b. and 10d.b.¼ five and ten days before the day of the estimationof LI.

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radiation (GR) 10 days before this same day of estimation of the

leaf injury (Table 3).

(b) Fumigation experiments. The PCA summarized 66.6% of

the total variability of the data from all the fumigation experi-

ments on the first two axes (46.1% on axis 1 and 20.5% on axis 2)

(Fig. 5). Leaf injury and SOD showed the highest correlation

with axis 1 (r ¼ �0.81 and �0.62, respectively). AA (r ¼ �0.57)

and POD (r ¼ �0.59) showed the strongest correlation with axis

2. In general, the sample units of the fumigation experiments

performed with 40 ppb of O3 were grouped on the positive side of

axis 1 and were characterized by high concentrations of AA and

activity of POD and a low intensity of leaf injury. In contrast, the

sample units of the experiments performed with 80 ppb of O3

Fig. 5 PCA (principal component analysis) biplot of the biological

responses measured in plants of I. nil ‘Scarlet O’Hara’ exposed to 40, 50

and 80 ppb under fumigation conditions. AA: ascorbic acid; SOD:

superoxide dismutase; POD: peroxidase; INJ: percentage of leaf injury.

This journal is ª The Royal Society of Chemistry 2012

were grouped on the negative side of axis 1 and were marked by

low concentrations of AA and activity of POD and a high

intensity of leaf injury. The sample units from the experiments

performed with 50 ppb arose in an intermediate position on the

axis 1.

A significant positive and linear relationship was verified

between the daily average percentage of leaf injury in the fumi-

gated plants and the ozone concentrations expressed as daily

accumulated doses over 20 ppb (Table 3).

Discussion

The weather conditions recorded in 2006 at Ibirapuera Park were

atypical. Among other abnormalities, we single out the occur-

rence of the high intensity of the solar radiation during many

days in the field experiment performed in the winter. In contrast,

several cloudy or rainy days were observed during the spring.

These atypical weather conditions were reflected in the O3

concentrations. They were lower than the historical averages

observed in the region of study and higher than expected during

the winter experiment.33

Anyway, the plants of I. nil were generally exposed to more

enhanced accumulated doses of O3 at Ibirapuera Park than were

the plants of the same bioindicator cultivar exposed by Dafr�e-

Martinelli et al.21 in another public park of Sao Paulo, aiming to

determining their redox states under varying environmental

conditions. This may explain why more severe visible injury

appeared on the leaves of the plants used in this study (chloroses

and necroses), compared to the leaf injury identified by Dafr�e-

Martinelli et al.21 in their plants (chloroses in the majority of

the cases). This comparison is one first indication that the

leaf injuries observed in the plants of both studies may be

attributed to O3.

The appearance of the leaf injury is also of practical interest to

critically analyze the potential of sensitive plant species for O3

biomonitoring in the natural environment. I. nil ‘Scarlet O’Hara’

showed typical interveinal chloroses and necroses under the

levels of atmospheric contamination by O3 observed during the

field experiments of the present study, as already defined by

Nouchi and Aoki20 under fumigation conditions. The leaf injury

in the field was similar to that observed during the fumigation

experiments performed under the realistic levels of atmospheric

contamination at Ibirapuera Park. This similarity confirms that

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the bioindicator characteristic of such a cultivar did not change

during the last thirty years by natural genetic variations or

eventual genetic improvement made by man for commercial

purposes. Additionally, the intensity of damage was propor-

tional to the level of atmospheric contamination in the field, as

contrasting percentages of leaf injury during the summer and

autumn experiments coincided with similar contrasting accu-

mulated doses of O3. Therefore, it may be employed for indi-

cating at least qualitatively the occurrence of toxic levels of O3 in

the sub-tropical region.

However, it is also important to verify if the varying O3

concentrations may significantly explain the varying intensities

of visible leaf injury. This critical question runs necessarily into

the discussion of the level of tolerance to oxidative stress imposed

by O3 and meteorological factors under field conditions.4,34–36

Dafr�e-Martinelli et al.21 already concluded that the bioindicator

efficiency of this cultivar exposed in locations of Sao Paulo

contaminated by low levels of O3 may be restricted by the

increased tolerance to oxidative stress, mainly conferred by

ascorbic acid and glutathione. They affirmed that this increased

tolerance has not prevented the sporadic occurrence of inter-

veinal chlorosis in leaves. However, it restricts the practical use

for routine biomonitoring, because such a kind of injury is

difficult to be visually analysed.

The present study confirmed that the antioxidative responses

of I. nil were seasonally marked, suggesting the occurrence of

different levels of sensitivity to O3 over the study under natural

and even fumigation conditions. The high values of antioxidants

found in the spring experiment performed in the field may reflect

the ideal environmental conditions for the growth of this

cultivar. Meteorological conditions during that period may have

promoted high rates of photosynthesis and possibly a high influx

of O3 in the plants. Consequently, the formation of ROS should

have been intensified, demanding an increased antioxidant

response. These seasonal oxidation–reduction variations,

affecting the level of tolerance against oxidative stress imposed

by O3, were also noted by many other authors.4,30,37–41

In fact, the canonical correspondence analysis pointed out the

significant relationship between the biological responses of I. nil

(the antioxidants and visible injury) and the varying environ-

mental conditions (air pollutants and climatic variables) as

a whole. Moreover, the multivariate analyses identified which

environmental factors were the most important to explain the

oscillations on the levels of the antioxidants measured.

O3 proved to be a key environmental factor that affected the

antioxidant responses in plants of I. nil. Increasing concentrations

of leaf AA and activity of POD, in the field experiment, were

consequences of the plant exposure to varying andmoderate levels

of the pollutant, as also observed by Dafr�e-Martinelli et al.21 In

contrast, decreasing levels of AA, under ozone fumigation

conditions, resulted from plant exposure to a more acute dose of

O3, which appears to indicate the increasing loss of antioxidative

capacity in the plants. This reduction in the content of AA was

a more conservative response to O3 than the reduction in the

enzymatic activity, which was influenced by distinct air tempera-

ture regimes during the fumigation experiment. Ascorbic acid has

really been indicated as one of the most important antioxidants

and a key indicator of environmental stresses in several plant

species.1,35,36,38,42,43 Numerous studies have observed that the

1966 | J. Environ. Monit., 2012, 14, 1959–1967

activity of SOD, POD and other enzymatic antioxidants can

oscillate in plants grown in an oxidative environment,1,44,45

depending on the stage of development and/or intrinsic metabolic

rhythms46,47 interfering with their responses to O3.

The multivariate analyses also showed clear changes (either

positive or negative) in the antioxidant levels in the plants of I. nil

‘Scarlet O’Hara’, exposed under the field conditions, throughout

the seasons, in response to short-term variations in the meteo-

rological variables (relative humidity, temperature, global radi-

ation and wind speed). The levels of antioxidants in these plants

might have also reflected the abnormality of meteorological

conditions during the year of study. This fact explains the

oscillations observed in the levels of antioxidants measured in the

plants sampled every week during each seasonal field experiment.

Some other authors also observed seasonality in antioxidants

associated with the meteorological characteristics of each season

of the year. Gilham and Dodge,48 for example, showed that the

levels of ascorbate, ascorbate-peroxidase and glutathione-

reductase in leaves of Pisum satium (L.) were lower during winter

and higher during summer. Burkey et al.38 also found a marked

seasonality in the levels of ascorbic acid in the redox state in

ozone-sensitive wildflowers in the Great Smoky Mountains

National Park (U.S.A.).

The multilinear regression analyses indicated that the rela-

tionships between the antioxidant responses of plants in the field

experiments and environmental factors were time-dependent, as

also revealed by Dafr�e-Martinelli et al.21 for the same bio-

indicator cultivar. Esposito et al.17 observed similar responses in

plants ofNicotiana tabacum ‘‘Bel-W3’’ exposed to ambient ozone

at another highly contaminated site in the city of Sao Paulo.

In addition, the increase in the percentage of visible leaf injury

on leaves of I. nil ‘Scarlet O’Hara’ could not be directly associ-

ated with increased levels of O3 in the atmosphere in the field, as

clearly observed under fumigation conditions. A hierarchical

association occurred among environmental changes, antioxidant

responses and occurrence of visible injury. The enhancement of

air temperature and global radiation, which may either stimulate

the photosynthesis or induce oxidative stress, firstly caused an

increase of the levels of ROS, affecting the redox state of the

plants. The visible injury on the leaves appeared only after the

decrease of the tolerance against oxidative stress occurred, as

indicated by the negative relationship with AA and POD.

Therefore, it is not so notable that O3 has not been identified as

a significant parameter to linearly explain the intensity of leaf

injury in the multivariate model, even considering that increased

temperature and solar radiation could have enhanced the

gaseous exchange and then the uptake of O3. On the other hand,

higher levels of particulate material in contact with the leaf

surfaces can cause a reduction in the O3 uptake, among other

gases, due to stomata obliteration. This may explain why PM10

was associated negatively with leaf injury.

In brief, the results of the present study confirmed that I. nil

‘Scarlet O’Hara’ is sensitive to moderate levels of O3 and toler-

ates the short-term variations in the meteorological conditions

generally observed in the sub-tropics. But, the first occurrence

and the progression of typical leaf injury during the days of

exposure of plants of such a bioindicator in the field will depend

on how intense the oxidative power of the environment is. If so,

its efficiency for quantitative O3 biomonitoring in the sub-

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tropics, based on the analysis of leaf area affected by visible

injury, will be compromised. Its employment for qualitative O3

biomonitoring would be better recommended.

Conclusions

I. nil ‘Scarlet O’Hara’ showed typical interveinal chloroses and

necroses on the older leaves under the levels of atmospheric

contamination observed during the field experiments of the

present study, but only after the levels of antioxidants have

decreased. Seasonality in the antioxidant responses observed in

the plants was associated with meteorological variables and

ozone concentrations five days before leaf analyses. The highest

levels of antioxidants occurred during the spring. The increase in

the percentage of visible leaf injury could not be directly asso-

ciated with increased levels of ozone in the atmosphere in the

field. A hierarchical and time-dependent association occurred

among environmental changes that induce oxidative stress or

increase in the photosynthesis (temperature and solar radiation),

decrease in the antioxidant responses (AA and POD) and finally

the occurrence of visible injury on the leaves.

Therefore, the efficiency of I. nil ‘Scarlet O’Hara’ for quanti-

tative O3 biomonitoring may be compromised in the sub-tropics,

depending on how intense the oxidative power of the environment

is. It is more appropriate for qualitative biomonitoring purposes.

Acknowledgements

The authors gratefully acknowledge FAPESP (Fundacao de

Amparo a Pesquisa no Estado de Sao Paulo; proc. 02/04751-6)

for the financial support, and CNPq (Conselho Nacional de

Desenvolvimento Cient�ıfico e Tecnol�ogico) for offering a MSc

scholarship to the first author. The manuscript was edited for

proper English language, grammar, punctuation, spelling, and

overall style by a highly qualified native English speaking editor

at American Journal Experts (www.journalexperts.com).

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