Journal of Atmospheric Chemistry (2006) 53: 185209

DOI: 10.10007/s10874-005-9006-6


C

Springer 2006

Calibration of Four Species of Tillandsia as Air

Pollution Biomonitors
EDUARDO D. WANNAZ and MARIA L. PIGNATA
Instituto Multidisciplinario de Biologia Vegetal (IMBIV), Consejo Nacional de Investigaciones
Cientificas y Tecnicas (CONICET). Catedra de Quimica General. Facultad de Ciencias Exactas,
Fisicas y Naturales. Universidad Nacional de Cordoba. Avda. Velez Sarsfield 1611. Ciudad
Universitaria (X5016 GCA) Cordoba, Argentina, e-mail: ewannaz@com.uncor.edu
pignata@com.uncor.edu.
(Received: 3 January 2005; accepted: 17 September 2005)
Abstract. Many organisms have been used as bioindicators of atmospheric contamination, with
moss and lichen species being the most common. However, studies using epiphytic vascular species
of Tillandsia have shown a good correlation between the presence of pollutants and the bioindicators
response. Therefore, the aim of our investigation was to calibrate and compare the response of four
Tillandsia species of Argentina to ascertain whether they could be used as atmospheric contamination
biomonitors. For this, we analysed the correlation between the levels of heavy metals in total atmospheric deposition samples and: a) their rate of enrichment; b) the physiological response of the plant
samples.
Tillandsia samples collected from a non contaminated area in the province of Cordoba were transplanted to four areas in the capital city with different sources of pollution (industrial or traffic emissions). They were exposed for a period of 3 to 6 months after which the concentrations of Pb, Ni, Fe, Zn,
Cu and S as well as the physiological parameters of foliar damage were determined. Simultaneously
samples of total atmospheric deposition were also taken.
The highest level of metal enrichment was found in T. capillaris followed by T. tricholepis, T.
permutata and T. retorta. Also, the use of a foliar damage index proved to be effective and could be a
useful tool to evaluate different levels of atmospheric quality in these species. The rate of heavy metal
deposition was higher in the industrial area for all metals except for Zn whose values were higher in
areas with high levels of traffic.
Key words: air pollution; atmospheric total deposition, biomonitors; heavy metals; Tillandsia.

1. Introduction
Biomonitoring is a simple and relatively inexpensive tool for prolonged surveys of
environmental quality because it provides the integrated effect of all the environmental factors including air pollution and weather conditions. Furthermore, when
biomonitoring is combined with physiochemical monitoring, the relationship between the concentration of a pollutant and its effects on the plants can be traced
(Chaphebar, 2000). Some epiphytic plants of the Tillandsia genus are efficient air
pollution biomonitors as they are not in contact with the soil, as their source of

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E. D. WANNAZ AND M. L. PIGNATA

water and nutrients is atmospheric. For this reason, their elemental tissue content
largely reflects the atmospheric levels of some toxic elements (Figueiredo et al.,
2001). T. usneoides has proven to efficiently accumulate atmospheric Hg present
in the neighbouring area of a chlor-alkali plant in Rio de Janeiro (Brazil) (Calasans
and Malm, 1997), as well as in urban areas (Malm et al., 1998). T. aeranthos and
T. recurvata have been used in Porto Alegre (Brazil) to determine the atmospheric
content of sulphur and heavy metals in both industrialized and residential areas
(Flores, 1987). In Colombia, the airborne heavy metal deposition in the highly
industrialized Cauca Valley was studied using T. recurvata as an accumulative
indicator (Schrimpf, 1984).
However, in order to establish and use epiphytic plants as air quality biomonitors
it is important to characterize the response of each species to different environmental
conditions, as well as to different levels of pollutants. Many species of Tillandsia
grow in a large area of Argentina; among them, T. capillaris has been used in a
preliminary study as a passive biomonitor of atmospheric contamination (Pignata
et al., 2002). We therefore considered that T. capillaris, T. permutata, T. tricholepis
and T. retorta, species that are abundant in the province of Cordoba, could be used
to detect the emission sources of heavy metals and to establish different levels of
air quality via active biomonitoring studies.
The aims of the present investigation were to: (a) calibrate the heavy metal accumulation in relation to total atmospheric deposition of four species of Tillandsia,
in order to be used as biomonitors and, (b) study the physiological response of each
species to air pollutants.

2. Materials and Methods

2.1.

STUDY AREA

The present study was undertaken in the city of Cordoba, located in the centre of
the Republic of Argentina (31 24 S, 64 11 W) at an altitude of approximately
400 m above sea level (Figure 1). It has a population of around 1.5 million and
an irregular topography. Its general structure is funnel-shaped, with an increasing
positive slope from the centre towards the surrounding areas. This somewhat concave formation reduces air circulation and causes frequent thermal inversions in
both autumn and winter. The climate is subhumid, with an average annual rainfall
of 790 mm, concentrated mainly in summer. The mean annual temperature is of
17.4 C and the prevailing winds come from the NE, S and SE.
The main source of air pollution in Cordoba city (downtown) is from mobile
sources (Stein and Toselli, 1996). The traffic pattern is closely related to all the primary pollutants measured (CO, NOx and PM10 ) which is another reason further justifying the assumption that the citys pollution is mainly due to mobile sources (Olcese and Toselli, 2002). Cordoba city also has an important industrial development
of mainly metallurgic and mechanical industries that are located in peripheral areas.

TILLANDSIA AS AIR POLLUTION BIOMONITORS

187

Figure 1. Location of the four areas in the city of Cordoba, Argentina, where the biomonitors
were transplanted and where the samples of total atmospheric deposition were taken().

2.2. B IOLOGICAL

MATERIAL , PLANT TRANSPLANTATION AND TOTAL

DEPOSITION SAMPLES

Tillandsia capillaris Ruz & Pav. f. capillaris, Tillandsia permutata A. Cast.,

Tillandsia tricholepis Baker and Tillandsia retorta Griseb. ex Baker, were collected from the Dique la Quebrada, a natural reserve considered an unpolluted
site found in the north of the province of Cordoba at approximately 40 km from the
capital city.
Plant bags containing 200 g plant material were prepared according to Gonzalez
and Pignata (1994) and transplanted simultaneously to four areas in the city of
Cordoba with different conditions of atmospheric contamination. In each area six
bags were bags were hung 3 m above ground level (n = 6 bags/species/site). The
process of transplantation was held on the 14th of June 2002 and corresponds to
the winter season in which the lowest rainfall values and temperatures are recorded

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E. D. WANNAZ AND M. L. PIGNATA

Table I. Mean values of rainfall and temperature in the

city of Cordoba during the months of June to December
2002
Month

Rainfall (mm)

T (Min-Max) ( C)

June
July
August
September
October
November
December

0.0
26.5
23.0
25.5
32.0
259.5
117.0

8.4 (1.1 15.7)

9.2 (2.2 16.2)
11.7 (3.9 19.6)
13.7 (5.2 22.2)
18.9 (11.3 26.5)
21.6 (14.4 29.9)
22.3 (16.6 28.0)

(Table I). Three and six months later (14th of September and 14th of December),
three bags of each species were recovered from each sampling site. The Tillandsia
samples were conserved at 15 C in the dark until the corresponding chemical
analyses were undertaken. Three assays were taken for each sample in every determination.
In order to establish the initial state of the samples before transplantation a
basal sample from the original collection site was analysed for each species. These
samples were used as the controls to which the transplanted samples were compared
to evaluate the changes in metal concentration and the physiological responses of
each species exposed to atmospheric contamination.
For the evaluation of the deposit flux, suitable experimental devices were adapted
from those described previously (Harrison and Johnston, 1985; Hewitt and Rashed,
1991) to collect wet and dry deposition. The device consisted of a polypropylene bottle with a funnel (20 cm diameter) that was located 3 m above ground
level in order to avoid soil interferences and was covered with a PVC laminated
net to prevent the entrance of any undesired objects (insects, leaves). The devices
were hung on the same day the plant material was transplanted. Half the devices
were collected 3 months later (n = 2 bottles/site) and the rest (n = 2 bottle/site)
after 6 months, on the same days the plant samples were collected. Before being exposed, the devises, they were washed with an acidified solution (HNO3 ,
5% V/V).
2.3.

TRANSPLANTATION AREAS

Four areas of the city of Cordoba were chosen for transplantation (Figure 1). Two
of these sites corresponded to highly contaminated places, one by traffic (Centre)
and the other by industrial activities (Southeast). The other two corresponded to
residential areas where high levels of atmospheric contamination are not expected.

TILLANDSIA AS AIR POLLUTION BIOMONITORS

189

Southeast area: an industrial area, with metallurgic and metal-mechanic

industries. The sampling site was located inside the premises of an industrial plant
in which metallic parts are made (tanks, radiators, covers, etc).
Centre area: the site was located downtown in a densely populated area. It is
where most of the public buildings, government offices, shops and businesses are
located and almost all the local buses run through it. The centre of the city is found
in a topographic depression and therefore the dispersion of the contaminants is
difficult.
North area: a suburban residential area in which houses and small and medium
shops are found. Traffic is much less than the centre area and is mainly composed
of cars with some public transport in the main avenues.
Southwest area: also a residential area with similar characteristics to the North
site, although the level of traffic is even less and there are many unpaved roads.

2.4.

ELEMENTAL ANALYSIS OF THE PLANT SAMPLES

The concentrations of Mn, Fe, Co, Ni, Cu, Zn and Pb were analysed in leaves of
Tillandsia (3g dry weight) taken from the plant samples that had been transplanted
for 3 and 6 months in each area as well as in leaves from non-transplanted plants
(basal state). The plant material was ground and reduced to ashes at 500 C for
4 hours. The ashes were digested with HCl (18%):HNO3 (3:1), the solid residue
was separated by centrifugation and the volume was adjusted to 25 mL with Milli-Q
water. After this, 10 ppm of a Ge solution was added as an internal standard. Aliquots
of 5 L were taken from this solution and dried on an acrylic support. As a quality
control, blanks and samples of the standard reference material Hay IAEA-V-10
were prepared in the same way. Standard solutions with known concentrations of
different elements and Ge as an internal standard were prepared for the calibration
of the system.
The samples were measured for 200 s, using the total reflection set up mounted
at the X-ray fluorescence beamline of the National Synchrotron Light Laboratory
(LNLS), Campinas, Brazil. For the excitation, a polychromatic beam was used
of approximately 0.3 mm wide and 2 mm high. For the X-ray detection, a Si(Li)
detector was used with an energy resolution of 165 eV at 5.9 keV.

2.5.

ELEMENTAL ANALYSIS OF THE TOTAL DEPOSITION SAMPLES

The total deposition samples (wet plus dry) of each corresponding area (3 and 6
months after exposure) were evaporated until dry and the residue was heated in
an oven at 500 C for 4 hours. The ashes were treated in the same way as those of
the plant samples in order to prepare the metallic acid solutions to be analysed by
TRXF. Results are expressed as g/m2 .exposure time.

190
2.6.

E. D. WANNAZ AND M. L. PIGNATA

DRY WEIGHT / FRESH WEIGHT RATIO

The dry weight/fresh weight ratio (DW/FW) of the samples was determined by
drying 4 g fresh material at 60 2 C until reaching constant weight. The results
were expressed in g DW. g1 FW.

2.7.

CHLOROPHYLL CONTENT

Chlorophyll extracts were prepared from 200 mg of plant material that was homogenised in 10 mL EtOH 96 % V/V with an Ultra Turrax homogeniser and the
supernatant was then separated by centrifugation. In order to measure phaeophytin
formation, 0.06 M HCl was added to clear the chlorophyll extract (1 mL HCl and 5
mL chlorophyll extract). Absorption of chlorophylls and phaeophytins, and that of
phaeophytins alone (after HCl addition) was measured with a Beckman DU 7000
spectrophotometer. Concentrations of chlorophylls and phaeophytins were calculated on a fresh weight basis (Pignata et al., 2002). The chlorophyll b/ chlorophyll
a (Chl-b /Chl-a) ratios were also calculated. The results are expressed in mg.g1
FW.

2.8.

SULPHUR CONTENT

Five millilitres of a Mg (NO3 )2 saturated solution were added to 0.5 g plant material
and dried in an electric heater. Subsequently, the sample was heated in an oven for 30
min at 500 C. The ashes were then suspended in 6 M HCl, filtered and the resulting
solution was boiled for 3 minutes. Finally the solution was brought to 50 mL
with distilled water.
The amount of SO2
4 in the solution was determined by the acidic suspension
method with BaCl2 which subsequently allowed to calculate the sulphur content
of each sample (Gonzalez and Pignata, 1994). The concentration was expressed in
mg of total sulphur.g1 FW.

2.9.

ESTIMATION OF PEROXIDATION PRODUCTS

The malondialdehyde (MDA) content was measured by a colorimetric method. The

amount of MDA present was calculated using = 155 mM1 cm1 (Kosugi et al.,
1989). The results were expressed in nmol g1 FW.
Hydroperoxy conjugated dienes (HPCD) were extracted by homogenization of
the plant material in 96% V/V EtOH at a ratio of 1:50 FW/V. The absorption was
measured in the supernatant at 234 nm and its concentration was calculated using
= 2.65 104 M1 cm1 (Levin and Pignata, 1995). The results were expressed
as mol g1 FW.

TILLANDSIA AS AIR POLLUTION BIOMONITORS

2.10.

191

FOLIAR DAMAGE INDEX

A Foliar Damage Index (FDI) was calculated in order to combine the changes of
each individual parameter measured indicating stress due to atmospheric pollution
(Pignata et al., 2002), according to the following equation:
FDI = [(Chl-b/Chl-a) + (S/Sb )] [(MDA/MDAb )
+ (HPCD/HPCDb )] (DW/FW)
where Chl-b is the chorophyll b concentration in mg g1 FW; Chl-a is the chorophyll
a concentration in mg g1 FW; S is the sulphur content in mg 1 FW; MDA is the
malondialdehyde concentration in nmol g1 FW; HPCD is the hydroperoxy conjugated dienes in mol g1 FW. The parameter with subindex b in the denominator
represents the arithmetic mean values calculated in the basal samples.
The FDI was used to compare the physiological response of the four studied
species to the presence of air pollutants and therefore the values were related to the
different levels of air quality.
2.11.

STATISTICAL ANALYSIS

Results are expressed as the mean values of three independent determinations for
each species in each sampling area. The data was analysed using a paired t-Test of
ANOVA and a value of p < 0.05 was considered a significant difference.
3. Results and Discussion
3.1.

HEAVY METAL CONTENT IN THE TOTAL DEPOSITION SAMPLES

Table II shows the mean concentration of metals in the total deposition samples
for each sampling area after 3 and 6 months exposure. For both periods, the concentration of Mn, Fe, Ni, Cu and Pb was significantly higher in the industrial area
(Southeast). The deposition of Zn was significantly higher in the Centre area. In
the samples corresponding to the 6 month period the concentrations of Co were
significantly higher in the Centre and Southeast areas than the North and Southwest
areas. Therefore, highest levels of metal deposition in the industrial area correspond
to the emissions of the metallurgic and metal-mechanic industries present in the
area.
On the other hand, the highest levels of Zn deposition were related to traffic
emission as they corresponded to the Centre area. High levels of Zn have been
reported for other areas in which the dispersion of traffic emission contaminants is
influenced by topographical characteristics and meteorological conditions (Viard
el at., 2004). Due to the fact that traffic pollutants include toxic metals such as
lead, cadmium and zinc (Caussy et al., 2003), these elevated levels of Zn could
have adverse effects on the populations health. After 6 months deposition elevated

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E. D. WANNAZ AND M. L. PIGNATA

Table II. Mean values (S.D.) of the amount of metal found in total atmospheric deposition
samples after 3 and 6 months transplantation, including the results of the ANOVA among
different areas
Deposition (g/m2 after 3 months)
Centre
Mean S.D.
Mn
Fe
Co
Ni
Cu
Zn
Pb

6.09 1.13 b
409.58 66.60 b
18.42 3.35 ab
3.49 0.34 b
9.75 0.84 b
797.33 41.29 a
6.60 1.55 b

Southeast
Mean S.D.

North
Mean S.D.

Southwest
Mean S.D.

18.00 2.51 a
1573.20 528.96 a
27.72 9.20 a
7.72 0.23 a
29.71 1.11 a
28.69 0.35 b
18.35 4.58 a

4.29 1.53 b
236.08 117.22 b
3.85 1.31 b
2.92 0.33 b
1.96 0.86 c
12.39 2.70 b
2.76 0.52 b

7.48 1.46 b
422.13 118.30 b
6.00 1.02 b
3.58 0.87 b
2.99 1.34 c
10.77 2.99 b
2.17 0.50 b

19.98 4.19 ab
688.97 194.21 b
12.01 1.93 b
5.90 0.18 bc
4.53 0.06 bc
20.51 1.11 b
3.71 1.06 b

ANOVA

Deposition (g/m2 after 6 months)

Mn
Fe
Co
Ni
Cu
Zn
Pb

8.14 0.37 b
647.84 40.75 b
50.82 2.35 a
10.26 0.60 ab
25.40 1.77 b
3965.01 643.38 a
14.43 1.36 b

31.40 3.85 a
2762.84 259.61 a
54.98 2.61 a
14.67 1.95 a
59.06 10.51 a
98.98 35.44 b
34.08 7.31 a

11.54 2.65 b
536.83 293.43 b
11.03 1.65 b
4.24 1.67 c
2.96 0.57 c
42.42 11.64 b
6.84 1.98 b

ns. not significant; significant with P < 0.05; significant with P < 0.01; significant with
P < 0.001. For each metal, values in the rows with the same letter do not differ significantly.
Post-hoc test: Tukey.

levels of Ni and Co were also found for the Centre area. Taking into account these
results, and as these are the first for Cordoba city regarding the emission of heavy
metals, we believe that further research is necessary to determine their atmospheric
levels as there is currently no organism monitoring the atmospheric contamination
of this city.
Viard et al. (2004) have informed that in an area near a highway in France the
amount of metal deposition was in decreasing order [Zn] > [Pb] > [Cd] in g
m2 d1 . It is therefore possible that these metals are enriched in urban soils and
so become dispersed as particulate atmospheric matter downtown. The elevated
levels of Zn we observed, in addition to descriptions for other areas of the world,
allow us to alert of the potential risk that this situation represents for the exposed
populations health. Urban soils are known to have peculiar characteristics such
as poor structure and high concentrations of trace elements (Tiller, 1992). Thus,
they are recipients of large amounts of heavy metals from a variety of sources
including vehicle emissions, cool burning wastes and other activities (Manta et al.,
2002).

TILLANDSIA AS AIR POLLUTION BIOMONITORS

3.2.

193

HEAVY METAL ENRICHMENT IN TILLANDSIA SPECIES

The mean values of metal concentration in the basal and transplanted samples
(3 and 6 months) of T. capillaris, T. permutata, T. tricholepis and T. retorta in each
area (Centre, Southeast, Southwest and North) are shown in Tables III, IV, V and
VI respectively.
In T. capillaris (Table III) the concentration of all metals increased after 3
and 6 months of exposure in all the study areas, with maximal enrichment values
for the Southeast area (industrial). This was observed even for Zn, although this
metal had the greatest deposition rates in the Centre area. The lowest values of
metal concentration corresponded to the samples transplanted to the residential
areas (North and Southwest) in accordance with the values of total atmospheric
deposition. After 3 months exposure some metals showed significant differences
compared to the basal samples, although there were no differences between the
sampling areas. We can therefore conclude that 3 months is not an adequate time
of exposure for the use of T. capillaris as an accumulation biomonitor.
A greater enrichment of Zn was found in T. permutata (Table IV) in all the study
areas after 6 months exposure. No significant differences were found for Mn with
respect to the basal values for both exposure periods. On the other hand, the highest
concentrations of Co, Cu and Pb were observed in the Southeast area (industrial)
after 6 months, according to the analysis of variance between areas.
Even though after 6 months exposure almost all the metals were enriched in
T. tricholepis (Table V) compared to the basal samples, this species only allowed to
difference the levels of Cu, Zn and Pb between the sampling areas, with the highest
levels corresponding to the Southeast and Centre areas after 6 months exposure.
After only 3 months exposure the concentrations of Zn and Cu in T. retorta
(Table VI) allowed to difference the studied areas, with the Southeast area being the site with the highest accumulation of Cu and the Southwest area the one
with the highest accumulation of Zn. After 6 months transplantation, all the metal
concentrations were increased with respect to the basal values. Thus, the highest
concentrations of Mn, Fe, Co and Cu were found in the industrial area (Southeast),
those of Pb in both the Southeast and Centre area and those of Zn in Centre and
Southwest sites.
Regarding the metal content of the biomonitors, the only two species that allowed
differencing the level of Mn accumulation between the study areas, and only after
6 months exposure, were T. capillaris and T. retorta. The Fe content in the transplanted samples increased in all the species and in almost all the areas compared to
the basal samples. However, the accumulation of this metal was only significant between the areas for T. capillaris, T. permutata y T. retorta and reflected the pattern of
deposition measured for this element. The pattern of deposition for Co was similar
to that of Fe and Mn and T. capillaris and T. retorta were the species that allowed
to discriminate the variation of this metal between the sampling areas. Total Co

194

E. D. WANNAZ AND M. L. PIGNATA

Table III. Comparison of the mean values of heavy metal concentrations (S.D.) measured
in T. capillaris. The values represent the result of a t-Test between the baseline sample and
the transplanted samples (3 and 6 months exposure) and the ANOVA between the different
transplantation areas
Mean SD
Centre

Southeast

North

Southwest

Mn
Baseline
3 months
6 months
t-Testa

13.579 0.277
24.872 3.016
58.107 5.349 b
(0 = 3 ; 0 = 6 )

13.579 0.277
16.187 4.663
85.052 5.290 a
(0 = 6 )

13.579 0.277
21.616 2.492
45.393 1.567 b
(0 = 3 ; 0 = 6 )

13.579 0.277
26.894 2.288
24.881 3.376 c
(0 = 3 ; 0 = 6 )

Fe
Baseline
3 months
6 months
t-Testa

197.04 38.63
291.14 47.71
1001.62 100.83 b
(0 = 6 )

197.04 38.63
269.17 75.67
1933.67 143.24 a
(0 = 6 )

197.04 38.63
312.76 13.31
879.52 139.59 b
(0 = 3 ; 0 = 6 )

197.04 38.63
287.93 34.723
503.97 27.86 c
(0 = 6 )

Co
Baseline
3 months
6 months
t-Testa

0.087 0.035
0.133 0.023
0.382 0.051 b
(0 = 6 )

0.087 0.035
0.117 0.030
0.725 0.089 a
(0 = 6 )

0.087 0.035
0.119 0.002
0.291 0.029 bc
(0 = 6 )

0.087 0.035
0.120 0.001
0.193 0.014 c
(0 = 6 )

Ni
Baseline
3 months
6 months
t-Testa

0.220 0.024
0.257 0.001
0.927 0.034 b
(0 = 6 )

0.220 0.024
0.362 0.095
1.796 0.132 a
(0 = 6 )

0.220 0.024
0.347 0.017
0.660 0.082 bc
(0 = 3 ; 0 = 6 )

0.220 0.024
0.348 0.069
0.416 0.081 c
(0 = 6 )

Cu
Baseline
3 months
6 months
t-Testa

1.249 0.142
1.591 0.151
5.007 0.075 b
(0 = 6 )

1.249 0.142
2.883 0.454
32.696 2.432 a
(0 = 3 ; 0 = 6 )

1.249 0.142
1.345 0.188
3.841 0.533 b
(0 = 6 )

1.249 0.142
2.260 0.618
2.051 0.182 b
(0 = 6 )

Zn
Baseline
3 months
6 months
t-Testa

4.003 0.126
7.326 1.306
19.806 0.815 b
(0 = 3 ; 0 = 6 )

4.003 0.126
7.259 2.822
47.248 4.447 a
(0 = 6 )

4.003 0.126
6.024 1.430
15.882 3.421 b
(0 = 6 )

4.003 0.126
6.883 0.472
6.551 0.594 c
(0 = 3 ; 0 = 6 )

Pb
Baseline
3 months
6 months
t-Testa

0.675 0.196
1.153 0.190
4.870 0.426 b
(0 = 6 )

0.675 0.196
1.244 0.092
13.945 0.231 a
(0 = 3 ; 0 = 6 )

0.675 0.196
0.613 0.291
3.675 0.251 b
(0 = 6 )

0.675 0.196
0.515 0.170
1.405 0.476 c
ns

ANOVAb

ns

ns

ns

ns

ns

ns

ns

ns, not significant; significant with P < 0.05; significant with P < 0.01; significant
with P < 0.001. a t-Test between Baseline (0) and exposure time; b ANOVA between transplantation areas. For each metal values in each row with the same letter do not differ significantly
(P < 0.05, Tukey pairwise comparison of means).

195

TILLANDSIA AS AIR POLLUTION BIOMONITORS

Table IV. Comparison of the mean values of heavy metal concentration (SD) measured in
T. permutata. The values represent the result of a t-Test between the baseline sample and
the transplanted samples (3 and 6 months exposure) and the ANOVA between the different
transplantation areas
Mean SD
Centre

Southeast

North

Southwest

ANOVAb

Mn
Baseline
3 months
6 months
t-Testa

20.927 1.946
20.111 1.188
30.559 10.513
ns

20.927 1.946
21.812 1.635
54.945 14.875
ns

20.927 1.946
13.791 8.083
45.767 14.094
ns

20.927 1.946
21.341 1.383
35.449 5.331
ns

ns
ns

Fe
Baseline
3 months
6 months
t-Testa

279.34 25.65
314.22 54.63
530.11 140.86
ns

279.34 25.65
308.18 4.02
1364.79 330.62
(0= 6 )

279.34 25.65
211.126 76.29
799.38 246.52
ns

279.34 25.65
271.41 13.63
679.77 93.31
(0= 6 )

ns
ns

Co
Baseline
3 months
6 months
t-Testa

0.115 0.004
0.137 0.022
0.202 0.064 b
ns

0.115 0.004
0.130 0.014
0.485 0.103 a
(0 = 6 )

0.115 0.004
0.100 0.044
0.309 0.007 ab
(0 = 6 )

0.115 0.004
0.117 0.004
0.213 0.017 b
(0 = 6 )

Ni
Baseline
3 months
6 months
t-Testa

0.379 0.023
0.397 0.060
0.593 0.091
ns

0.379 0.023
0.498 0.067
1.392 0.616
ns

0.379 0.023
0.315 0.186
0.766 0.007
(0 = 6 )

0.379 0.023
0.407 0.096
0.595 0.100
ns

Cu
Baseline
3 months
6 months
t-Testa

1.913 0.285
2.047 0.093 ab
4.112 1.572 b
ns

1.913 0.285
3.410 0.611 a
19.527 3.646 a
(0 = 6 )

1.913 0.285
1.354 0.571 b
3.433 0.831 b
ns

1.913 0.285
2.105 0.589 ab
3.487 0.527 b
ns

Zn
Baseline
3 months
6 months
t-Testa

12.597 0.251
30.072 1.182 a
41.779 8.255 ab
(0 3 ; 0 = 6 )

12.597 0.251
22.991 6.086 ab
42.612 9.006 ab
(0 = 6 )

12.597 0.251
20.117 4.307 ab
65.649 8.453 a
(0 = 6 )

12.597 0.251
12.693 1.844 b
17.851 1.100 b
(0 = 6 )

Pb
Baseline
3 months
6 months
t-Testa

0.812 0.112
0.473 0.117
1.741 0.346 b
ns

0.812 0.112
0.285 0.114
11.216 1.662 a
(0 = 3 ; 0 = 6 )

0.812 0.112
0.731 0.268
3.292 0.447 b
(0 = 6 )

0.812 0.112
0.367 0.056
3.398 1.511 b
(0 = 3 )

ns

ns
ns

ns

ns, not significant; significant with P < 0.05; significant with P < 0.01; significant with
P < 0.001. a t-Test between Baseline (0) and exposure time; b ANOVA between transplantation areas. For each metal values in each row with the same letter do not differ significantly
(P < 0.05, Tukey pairwise comparison of means).

196

E. D. WANNAZ AND M. L. PIGNATA

Table V. Comparison of the mean values of heavy metal concentration (SD) measured in
T. tricholepis. The values represent the result of a t-Test between the baseline sample and
the transplanted samples (3 and 6 months exposure) and the ANOVA between the different
transplantation areas
Mean SD

Mn
Baseline
3 months
6 months
t-Testa
Fe
Baseline
3 months
6 months
t-Testa
Co
Baseline
3 months
6 months
t-Testa
Ni
Baseline
3 months
6 months
t-Testa
Cu
Baseline
3 months
6 months
t-Testa
Zn
Baseline
3 months
6 months
t-Testa
Pb
Baseline
3 months
6 months
t-Testa

ANOVAb

Centre

Southeast

North

Southwest

16.192 1.345
26.529 0.437
45.175 1.570
(0 = 3 ; 0 = 6 )

16.192 1.345
27.455 1.024
58.932 24.38
(0 = 3 )

16.192 1.345
26.298 5.008
78.543 5.995
(0 = 6 )

16.192 1.345
18.298 0.923
46.291 16.265
ns

ns
ns

184.77 34.71
293.83 14.04
951.60 111.15
(0 = 6 )

184.77 34.71
344.53 56.47
1336.01 277.68
(0 = 6 )

184.77 34.71
357.39 50.67
1090.51 113.48
(0 = 6 )

184.77 34.71
209.08 3.09
819.65 391.40
ns

ns
ns

0.102 0.015
0.164 0.017
0.308 0.010
(0 = 6 )

0.102 0.015
0.139 0.033
0.546 0.078
(0 = 6 )

0.102 0.015
0.168 0.025
0.376 0.058
(0 = 6 )

0.102 0.015
0.096 0.028
0.275 0.119
ns

ns
ns

0.248 0.042
0.197 0.121
0.706 0.157
ns

0.248 0.042
0.234 0.048
1.080 0.140
(0 = 6 )

0.248 0.042
0.261 0.037
0.718 0.200
ns

0.248 0.042
0.209 0.026
0.467 0.054
(0 = 6 )

ns
ns

1.095 0.217
1.526 0.312
7.239 1.532 b
(0 = 6 )

1.095 0.217
2.835 0.729
21.425 2.312 a
(0 = 6 )

1.095 0.217
3.464 2.006
3.863 0.058 b
(0 = 6 )

1.095 0.217
0.931 0.272
2.600 0.316 b
(0 = 6 )

3.128 1.175
4.692 0.512
24.906 3.924 a
(0 = 6 )

3.128 1.175
5.339 1.641
20.723 4.797 ab
(0 = 6 )

3.128 1.175
5.868 1.369
10.891 1.575 b
(0 = 6 )

3.128 1.175
4.077 0.064
7.532 1.797 b
ns

0.318 0.124
0.510 0.089
1.701 0.010 ab
(0 = 6 )

0.318 0.124
0.769 0.169
3.858 1.071 a
(0 = 6 )

0.318 0.124
0.547 0.265
1.748 0.396 ab
(0 = 6 )

0.318 0.124
0.373 0.102
0.472 0.006 b
ns

ns

ns

ns

ns, not significant; significant with P < 0.05; significant with P < 0.01; significant with P < 0.001. a t-Test between Baseline (0) and exposure time; b ANOVA between
transplantation areas. For each metal values in each row with the same letter do not differ
significantly (P < 0.05, Tukey pairwise comparison of means).

197

TILLANDSIA AS AIR POLLUTION BIOMONITORS

Table VI. Comparison of the mean values of heavy metal concentration (SD) measured
in T. retorta. The values represent the result of a t-Test between the baseline sample and
the transplanted samples (3 and 6 months exposure) and the ANOVA between the different
transplantation areas
Mean SD

Mn
Baseline
3 months
6 months
t-Testa
Fe
Baseline
3 months
6 months
t-Testa
Co
Baseline
3 months
6 months
t-Testa
Ni
Baseline
3 months
6 months
t-Testa
Cu
Baseline
3 months
6 months
t-Testa
Zn
Baseline
3 months
6 months
t-Testa
Pb
Baseline
3 months
6 months
t-Testa

Centre

Southeast

North

Southwest

17.468 2.655
15.673 4.747
21.326 0.594 bc
ns

17.468 2.655
19.509 2.489
38.014 5.869 a
(0 = 6 )

17.468 2.655
26.781 1.735
31.841 0.578 ab
(0 = 3 ; 0 = 6 )

17.468 2.655
17.958 0.281
12.854 2.214 c
ns

155.01 12.56
138.95 51.15
288.64 71.61 b
(0 = 6 )

155.01 12.56
239.32 19.24
857.99 122.43 a
(0 = 3 ; 0 = 6 )

155.01 12.56
170.99 39.97
442.178 76.88 b
(0 = 6 )

155.01 12.56
169.86 19.62
152.24 34.56 b
ns

0.057 0.009
0.120 0.038
0.105 0.023 b
(0 = 6 )

0.057 0.009
0.092 0.015
0.301 0.040 a
(0 = 3 ; 0 = 6 )

0.057 0.009
0.113 0.048
0.134 0.011 b
(0 = 6 )

0.057 0.009
0.071 0.027
0.071 0.016 b
ns

0.454 0.024
0.402 0.083
0.574 0.068
ns

0.454 0.024
0.430 0.074
1.005 0.118
(0 = 6 )

0.454 0.024
0.565 0.136
0.882 0.176
(0 = 6 )

0.454 0.024
0.367 0.018
0.667 0.208
(0 = 3 )

1.397 0.108
2.043 0.342 ab
2.184 0.105 b
(0 = 3 ; 0 = 6 )

1.397 0.108
3.234 0.307 a
8.213 0.199 a
(0 = 3 ; 0 = 6 )

1.397 0.108
2.462 0.466 ab
2.051 0.171 b
(0 = 3 ; 0 = 6 )

1.397 0.108
1.535 0.060 b
1.321 0.174 c
ns

8.114 4.533
4.009 0.840 b
39.434 3.487 a
(0 = 6 )

8.114 4.533
5.635 1.067 b
13.264 0.965 c
ns

8.114 4.533
6.192 0.025 b
23.705 4.028 bc
(0 = 6 )

8.114 4.533
35.315 0.172 a
34.259 3.470 ab
(0 = 3 ; 0 = 6 )

0.363 0.127
0.409 0.060
0.695 0.102 ab
ns

0.363 0.127
0.703 0.099
1.656 0.297 a
(0 = 6 )

0.363 0.127
0.440 0.152
0.620 0.398 b
ns

0.363 0.127
0.369 0.136
0.538 0.021 b
ns

ANOVAb

ns

ns

ns

ns
ns

ns

ns, not significant; significant with P < 0.05; significant with P < 0.01; significant with
P < 0.001. a t-Test between Baseline (0) and exposure time; b ANOVA between transplantation areas. For each metal values in each row with the same letter do not differ significantly
(P < 0.05, Tukey pairwise comparison of means).

198

E. D. WANNAZ AND M. L. PIGNATA

deposition was greater in the Southeast and Centre areas and this was reflected in the
T. capillaris samples that were the ones that accumulated the highest concentrations
of this element.
Concerning Ni, T. capillaris was able to distinguish areas with different levels
of accumulation after 6 months exposure with a pattern that coincided with the total
deposition values measured.
The enrichment levels of Cu found in T. permutata and T. retorta differenced
the industrial area from the rest of the sites after only 3 months exposure, whereas
the rest of the species manifested this difference only after being transplanted for
6 months.
The levels of the concentration of Zn in samples transplanted for 6 months were
significantly higher than the basal samples for all species. However, whereas T.
capillaris showed differences between areas with the highest values belonging to
the Southeast site, in T. permutata the highest values corresponded to the North,
Centre and Southeast areas and T. tricholepis and T. retorta showed the highest
values for the Centre and Southeast areas. It is important to note that these two
last species showed a pattern of Zn accumulation coincident with the levels of total
deposition.
With respect to Pb, T. capillaris and T. permutata showed a similar behaviour
having the highest levels of enrichment in the Southeast (industrial) area in samples
exposed for 6 months. T. retorta had the highest concentrations of this metal in the
Southeast and Centre areas and T. tricholepis in the Southeast, Centre and North
sites. It is interesting to note that in all the species the highest concentrations of Pb
corresponded to the industrial site followed by the Centre site.

3.3.

ENRICHMENT FACTORS

In order to compare the capacity to accumulate metals of the different species, the
enrichment factors for each metal in every species were calculated according to the
following equation:
EF = X i j / X b
where X i j is the concentration of element X in mg g1 FW in time i and site
j; X b is the concentration of element X in mg g1 FW in the basal samples. The
maximal values of enrichment for each metal in each of the four species are shown in
Table VII.
Cu, Zn and Pb were the metals most enriched in T. capillaris, T. permutata
and T. tricholepis indicating their anthropogenic origin. Among these species,
T. capillaris had the highest accumulation of all the metals analysed thus exhibiting
a greater bioaccumulation capacity. On the contrary, T. retorta was the species that
showed the lowest levels of enrichment for all metals.

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TILLANDSIA AS AIR POLLUTION BIOMONITORS

Table VII. Maximal enrichment factors of the heavy metals
quantified in the four species of Tillandsia

Mn
Fe
Co
Ni
Cu
Zn
Pb

T. capillaris

T. permiutata

T. tricholepis

T. retorta

6.26
9.81
8.33
8.16
26.18
11.80
20.66

2.63
4.86
4.22
3.67
10.21
5.21
13.81

4.85
7.23
5.35
4.35
19.57
7.96
12.13

2.18
5.54
5.28
2.21
5.88
4.86
4.56

Table VIII. Lineal regression coefficients (R 2 ) and the parameter (a) for the function T s =
aTD + b where Ts is the elemental content of the Tillandsia species and TD the value of Total
Deposition
T. capillaris
R
Mn
Fe
Co
Ni
Cu
Zn
Pb

3.4.

0.27
0.55
0.64
0.79
0.76
0.02
0.77

T. permutala
2

1.33
0.51
0.009
0.11
0.46
0.001
0.37

0.50
0.53
0.38
0.55
0.63
0.06
0.57

T. tricholepis
2

1.21
0.35
0.004
0.07
0.26
0.003
0.26

0.17
0.27
0.38
0.58
0.76
0.45
0.70

T. retorta
2

0.98
0.27
0.005
0.06
0.29
0.004
0.09

0.16
0.66
0.36
0.27
0.82
0.19
0.79

a
0.39
0.24
0.002
0.03
0.10
0.004
0.04

significant with P 0.05, significant with P 0.01, significant with P 0.001.

LINEAL REGRESSION

In order to compare the ability to accumulate atmospheric metals of the four Tillandsia species, lineal regressions were made between the metal content of the total
deposition values and the concentration of metals found in the biomonitor species
(Table VIII). These were significant for all metals except for Zn in T. capillaris,
T. permutata and T. retorta and for Mn in T. tricholepis and T. retorta. Parameter
a, that represents the increase of the concentration of a determined metal in the
biomonitor that arises with the increase of one unit in the total deposition values
of the metal, was very low for Co and Zn in all the studied species indicating their
very low efficiency to accumulate these elements. On the other hand, the differences
between the concentrations of Zn and its deposition levels could be due to the fact
that this metal is possibly present in different chemical forms depending on the
source of emission from which it is originated (for example ionic forms, metallic
forms with an oxidation state of cero, organometallic compounds, etc.).

200

E. D. WANNAZ AND M. L. PIGNATA

Figure 2. Lineal regression analysis between the total deposition of Cu and its content in
T. capillaris (a), T. permutata (b), T. tricholepis (c) y T. retorta (d).

The other elements (Mn, Fe, Ni, Cu and Pb) showed the highest increase rates
in all the four species. Amongst them T. capillaris was the species with the highest
values of a, indicating a greater efficiency of accumulation as also shown for Cu
in Figure 2 and for Pb in Figure 3. As can be seen, differences in the efficiency of
accumulation of most metals were found among the studied species, being highest
in T. capillaris followed by T. permutata > T. tricholepis > T. retorta in decreasing
order. This efficiency could possibly be related to the anatomical features of each
species: for example, T. capillaris is the species with the greatest specific foliar
area (foliar area per gram of weight) and T. retorta the species with the smallest.
3.5.

PHYSIOLOGICAL PARAMETERS

In order to calibrate the physiological response of each species to the atmospheric

pollutants, the concentration of chemical physiological parameters was compared

TILLANDSIA AS AIR POLLUTION BIOMONITORS

201

Figure 3. Lineal regression analysis between the total deposition of Pb and its content in
T. capillaris (a), T. permutata (b), T. tricholepis (c) y T. retorta (d).

between both periods of transplantation for each species and for each area studied.
These parameters were chosen taking into account previous studies showing that
they were sensible to air pollution in other bioindicators such as lichens (Cuny
et al., 2002) and plants (Wannaz et al., 2003).
For T. capillaris (Table IX), the samples that evidenced the greatest physiological damage manifested by the increase in MDA and by the highest values of FDI,
were those exposed for 6 months in the Southeast and Centre areas. The levels of
sulphur were also greatest in these areas. It is important to note that the association
of the accumulation of sulphur in a biomonitor with lipid peroxidation in membranes is a clear indication of damage due to air pollution, as previously described
for lichens (Cuny et al., 2000). Furthermore, the content of sulphur in T. capillaris
leaves significantly increased after 3 months compared to the basal values and was
even higher after 6 months exposure. It is interesting to state that the sulphur content
was greater in the Southeast and Centre sites that are near emission sources of fossil
derived fuels.

202

E. D. WANNAZ AND M. L. PIGNATA

Table IX. Comparison of the mean values SD of the chemical parameters measured in
T. capillaris. The values represent the result of a t-Test between the baseline sample and
the transplanted samples (3 and 6 months exposure) and the ANOVA between the different
transplantation areas
Mean SD
Centre

Southeast

North

Southwest

Chl-a
Baseline
3 months
6 months
t-Testa

0.340 0.020
0.284 0.010 ab
0.328 0.009
ns

0.340 0.020
0.187 0.027 b
0.311 0.017
(0 = 3 )

0.340 0.020
0.222 0.018 ab
0.286 0.014
(0 = 3 )

0.340 0.020
0.303 0.045 a
0.279 0.015
ns

Chl-a/Chl-b
Baseline
3 months
6 months
t-Testa

0.416 0.009
0.460 0.016
0.557 0.071
ns

0.416 0.009
0.420 0.042
0.462 0.005
(0 = 6 )

0.416 0.009
0.416 0.029
0.485 0.060
ns

0.416 0.009
0.421 0.030
0.450 0.041
ns

MDA
Baseline
3 months
6 months
t-Testa

16.206 1.491
33.829 4.131
30.880 8.472 ab
(0 = 3 )

16.206 1.491
30.861 0.619
47.101 0.221 a
(0 = 3 ; 0 = 6 )

16.206 1.491
33.122 1.867
26.635 0.922 b
(0 = 3 ; 0 = 6 )

16.206 1.491
32.786 2.834
26.923 1.717 b
(0 = 3 ; 0 = 6 )

HPCD
Baseline
3 months
6 months
t-Testa

6.169 0.137
10.644 2.710
8.995 0.717
(0 = 6 )

6.169 0.137
10.578 1.693
9.247 2.054
ns

6.169 0.137
8.649 1.009
7.888 1.195
ns

6.169 0.137
8.939 1.791
7.261 2.621
ns

Sulphur
Baseline
3 months
6 months
t-Testa

0.155 0.137
0.312 0.005 a
0.568 0.001 a
(0 = 3 ; 0 = 6 )

0.155 0.137
0.241 0.013 b
0.634 0.064 a
(0 = 3 ; 0 = 6 )

0.155 0.137
0.212 0.009 b
0.361 0.046 b
(0 = 3 ; 0 = 6 )

0.155 0.137
0.216 0.014 b
0.290 0.025 b
(0 = 3 ; 0 = 6 )

DW/FW
Baseline
3 months
6 months
t-Testa

0.212 0.007
0.243 0.017
0.541 0.092
(0 = 3 ; 0 = 6 )

0.212 0.007
0.235 0.010
0.510 0.111
(0 = 3 ; 0 = 6 )

0.212 0.007
0.234 0.007
0.486 0.049
(0 = 3 ; 0 = 6 )

0.212 0.007
0.236 0.020
0.423 0.088
(0 = 6 )

FDI
Baseline
3 months
6 months
t-Testa

0.621 0.014
2.293 0.432
7.680 1.057 a
(0 = 3 )

0.621 0.014
1.596 0.242
10.255 1.739 a
(0 = 3 ; 0 = 6 )

0.621 0.014
1.481 0.048
2.788 0.664 b
(0 = 3 ; 0 = 6 )

0.621 0.014
1.437 0.048
4.005 0.850 b
(0 = 3 ; 0 6 )

ANOVAb

ns

ns
ns

ns

ns
ns

ns
ns

ns

ns, not significant; significant, P < 0.05; significant, P < 0.01; significant, P <
0.001. a t-Test between Baseline (0) and exposure time; b ANOVA between areas. For each
metal the values in the rows with the same letter do not differ significantly, P < 0.05 (Tukey,
pairwise comparison of means).

TILLANDSIA AS AIR POLLUTION BIOMONITORS

203

The FDI of T. capillaris divided the sampling areas into two groups. One group
included the Southeast and Centre areas, in which the species suffered greater
physiological damage; the other group included the Southwest and Northern areas
where the samples had a lower degree of physiological damage even after 6 months
exposure. As these results are in accordance to the results for the metal concentrations, we can say that T. capillaris is a good biomonitor in both its physiological
response and its heavy metal accumulation. Thus, the air pollution biomarkers for
this species are MDA, sulphur and the FDI.
Physiological damage was evidenced in the samples of T. permutata from the
industrial area (Southeast) as its levels of HPCD, DW/FW, sulphur and its FDI
increased (Table X). The value of the FDI after 6 months exposure was significantly
higher for the Southeast area and much less for the Centre area. Therefore, this
species is possibly tolerant to air contaminants released by traffic or more sensitive
to metals and sulphur emissions originated by industrial activity.
T. tricholepis (Table XI) showed an early increase (3 months exposure) in its
MDA levels in the Southeast (industrial) area and in its sulphur and FDI values
in all the transplantation areas. The values of the FDI were greater in the Centre
and Southeast areas after 3 months exposure and in the Southeast and Northern
area after 6 months compared to the basal values for these species. The biomarkers for this species were the sulphur content and the FDI, both allowing to difference areas with different levels of air pollution after a short period of exposure and also coinciding with the deposition pattern of the atmospheric particulate
matter.
The greatest physiological damage was observed in T. retorta (Table XII) according to its value of FDI and was associated to the air pollutants present in the
downtown area (traffic emission). On the other hand, the highest concentrations
of HPCD were found in the industrial area, those of sulphur in the Centre area
and the greatest DW/FW ratios were observed in both the Centre and Industrial
areas (Southeast). The biomarkers of damage by air pollution for this species were
sulphur, HPCD, DW/FW and FDI. Further studies are recommended for T. retorta
in order to confirm whether the physiological damage associated to the presence
of air pollutants prevents the accumulation of metals as this species presented the
lowest levels of enrichment.
It is important to point out that, even though the physiological responses of plants
strongly depend on climatic conditions (seasonal variations), this study provides
evidence that the differences in the physiological parameters of Tillandsia between
the sampling periods were not greater than the differences observed between the
areas with different levels of contamination.
The ratio of metal accumulation for almost all the species studied was higher
during the second period, coincident with the higher rates of total deposition for
most of the metals analysed in the most contaminated areas. Contrary to what was
expected by the increased levels of rainfall during the second period of exposure,

204

E. D. WANNAZ AND M. L. PIGNATA

Table X. Comparison of the mean values SD of the chemical parameters measured in T.

permutata. The values represent the result of a t-Test between the baseline sample and the
transplanted samples (3 and 6 months exposure) and the ANOVA between the different transplantation areas
Mean SD
Centre

Southeast

North

Southwest

ANOVAb

Chl-a
Baseline
3 months
6 months
t-Testa

0.297 0.024
0.268 0.093
0.352 0.059
ns

0.297 0.024
0.208 0.059
0.316 0.060
ns

0.297 0.024
0.320 0.013
0.195 0.015
(0 = 6 )

0.297 0.024
0.255 0.068
0.335 0.019
ns

ns
ns

Chl-b/Chl-a
Baseline
3 months
6 months
t-Testa

0.387 0.007
0.447 0.008
0.464 0.009
(0 = 3 ; 0 = 6 )

0.387 0.007
0.356 0.056
0.468 0.059
ns

0.387 0.007
0.480 0.022
0.478 0.013
(0 = 3 ; 0 = 6 )

0.387 0.007
0.410 0.020
0.433 0.054
ns

ns
ns

MDA
Baseline
3 months
6 months
t-Testa

16.567 1.533
27.088 1.305
25.809 5.079
(0 = 3 )

16.567 1.533
30.452 4.333
42.866 3.409
(0 = 6 )

16.567 1.533
25.668 7.778
35.682 4.511
(0 = 6 )

16.567 1.533
28.594 9.534
40.115 8.488
ns

ns
ns

HPCD
Baseline
3 months
6 months
t-Testa

8.159 0.236
10.654 0.768
6.741 0.574 b
(0 = 3 )

8.159 0.236
6.962 1.373
11.160 0.732 a
(0 = 6 )

8.159 0.236
7.524 0.174
5.319 1.150 b
ns

8.159 0.236
7.605 1.319
8.228 0.732 ab
ns

Sulphur
Baseline
3 months
6 months
t-Testa

0.247 0.002
0.275 0.008
0.332 0.012 b
(0 = 3 ; 0 = 6 )

0.247 0.002
0.253 0.033
0.739 0.104a
(0 = 6 )

0.247 0.002
0.283 0.034
0.445 0.053 b
(0 = 6 )

0.247 0.002
0.319 0.055
0.510 0.015 ab
(0 = 6 )

DW/FW
Baseline
3 months
6 months
t-Testa

0.221 0.010
0.211 0.011
0.253 0.022 b
ns

0.221 0.010
0.199 0.011
0.642 0.079 a
(0 = 6 )

0.221 0.010
0.206 0.011
0.397 0.047 b
(0 = 6 )

0.221 0.010
0.194 0.001
0.390 0.058 b
(0 = 6 )

FDI
Baseline
3 months
6 months
t-Testa

0.612 0.021
0.969 0.082
1.095 0.208 c
(0 = 3 )

0.612 0.021
0.739 0.076
8.751 0.560 a
(0 = 6 )

0.612 0.021
0.821 0.070
2.522 0.151 b
(0 = 6 )

0.612 0.021
0.889 0.263
3.331 0.433 b
(0 = 6 )

ns

ns

ns

ns

ns, not significant; significant, P < 0.05; significant, P < 0.01; significant, P < 0.001.
a
t-Test between Baseline (0) and exposure time; b ANOVA between areas. For each metal the
values in the rows with the same letter do not differ significantly, P < 0.05 (Tukey pairwise
comparison of means).

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TILLANDSIA AS AIR POLLUTION BIOMONITORS

Table XI. Comparison of the mean values SD of the chemical parameters measured in
T. tricholepis. The values represent the result of a t-Test between the baseline sample and
the transplanted samples (3 and 6 months exposure) and the ANOVA between the different
transplantation areas
Mean SD
Centre

Southeast

North

Southwest

ANOVAb

0.265 0.015
0.249 0.066
0.314 0.055
ns

0.265 0.015
0.339 0.068
0.385 0.063
ns

0.265 0.015
0.365 0.135
0.349 0.057
ns

0.265 0.015
0.230 0.058
0.307 0.028
ns

ns
ns

Chl-a/Chl-b
Baseline
0.475 0.098
3 months
0.547 0.137
6 months
0.492 0.036
ns
t-Testa

0.475 0.098
0.487 0.065
0.491 0.014
ns

0.475 0.098
0.485 0.007
0.508 0.001
ns

0.475 0.098
0.449 0.014
0.448 0.022
ns

ns
ns

MDA
Baseline
3 months
6 months
t-Testa

12.611 0.056
19.974 2.757
18.825 0.396
(0 = 6 )

12.611 0.056
26.984 4.660
20.517 0.627
(0 = 3 0 = 6 )

12.611 0.056
17.007 5.787
20.981 5.035
ns

12.611 0.056
17.998 2.907
18.275 3.516
ns

ns
ns

HPCD
Baseline
3 months
6 months
t-Testa

10.321 0.541
10.398 0.284
7.375 1.475
ns

10.321 0.541
9.977 0.012
8.603 0.551
ns

10.321 0.541
10.828 6.716
8.405 3.408
ns

10.321 0.541
6.767 2.368
6.507 2.098
ns

ns
ns

Sulphur
Baseline
3 months
6 months
t-Testa

0.114 0.004
0.197 0.002
0.489 0.073 a
(0 = 3 ; 0 = 6 )

0.114 0.004
0.182 0.002
0.542 0.007 a
(0 = 3 ; 0 = 6 )

0.114 0.004
0.181 0.026
0.435 0.006 ab
(0 = 3 ; 0 = 6 )

0.114 0.004
0.178 0.005
ns

0.304 0.037 b
(0 = 3 ; 0 = 6 )

DW/FW
Baseline
3 months
6 months
t-Testa

0.157 0.006
0.177 0.007
0.327 0.071
(0 = 3 ; 0 = 6 )

0.157 0.006
0.178 0.012
0.451 0.054
(0 = 6 )

0.157 0.006
0.169 0.006
0.511 0.191
(0 = 6 )

0.157 0.006
0.160 0.007
0.293 0.005
(0 = 6 )

FDI
Baseline
3 months
6 months
t-Testa

0.448 0.038
1.087 0.109 a
3.388 0.092 b
(0 = 3 ; 0 = 6 )

0.448 0.038
1.127 0.080 a
5.826 0.62l a
(0 = 3 ; 0 = 6 )

0.448 0.038
0.822 0.024 b
5.174 0.373 a
(0 = 3 ; 0 = 6 )

0.448 0.038
0.653 0.033 b
1.893 0.152 c
(0 = 3 ; 0 = 6 )

Chl-a
Baseline
3 months
6 months
t-Testa

ns
ns

ns, not significant; significant, P < 0.05; significant, P < 0.01; significant, P < 0.001.
a
t-Test between Baseline (0) and exposure time; b ANOVA between areas. For each metal the
values in the rows with the same letter do not differ significantly, P < 0.05 (Tukey pairwise
comparison of means).

206

E. D. WANNAZ AND M. L. PIGNATA

Table XII. Comparison of the mean values SD of the chemical parameters measured in T.
retorta. The values represent the result of a t-Test between the baseline sample and the transplanted samples (3 and 6 months exposure) and the ANOVA between the different transplantation areas
Mean SD
Centre

Southeast

North

Southwest

ANOVAb

Chl-a
Baseline
3 months
6 months
t-Testa

0.181 0.020
0.235 0.045
0.248 0.082
ns

0.181 0.020
0.214 0.003
0.240 0.055
ns

0.181 0.020
0.159 0.012
0.160 0.002
ns

0.181 0.020
0.177 0.019
0.176 0.008
ns

ns
ns

Chl-a/Chl-b
Baseline
3 months
6 months
t-Testa

0.391 0.064
0.430 0.073
0.462 0.040
ns

0.391 0.064
0.440 0.022
0.492 0.013
ns

0.391 0.064
0.367 0.096
0.367 0.095
ns

0.391 0.064
0.341 0.040
0.493 0.001
ns

ns
ns

MDA
Baseline
3 months
6 months
t-Testa

16.809 2.258
20.278 5.437
25.620 6.310
ns

16.809 2.258
26.365 5.818
17.279 2.563
ns

16.809 2.258
18.974 4.340
19.289 0.130
ns

16.809 2.258
14.006 1.462
15.438 0.825
ns

ns
ns

HPCD
Baseline
3 months
6 months
t-Testa

3.879 0.118
3.739 0.011
2.551 0.818 b
ns

3.879 0.118
6.115 1.243
7.946 2.092 a
ns

3.879 0.118
3.750 0.296
2.986 1.317 ab
ns

3.879 0.118
5.484 3.114
4.266 0.041 ab
ns

Sulphur
Baseline
3 months
6 months
t-Testa

0.098 0.009
0.144 0.007
0.411 0.011 a
(0 = 3 ; 0 = 6 )

0.098 0.009
0.147 0.009
0.231 0.012 b
(0 = 3 ; 0 = 6 )

0.098 0.009
0.163 0.012
0.183 0.004 b
(0 = 3 ; 0 = 6 )

0.098 0.009
0.155 0.011
0.195 0.054 b
(0 = 3 ; 0 = 6 )

DW/FW
Baseline
3 months
6 months
t-Testa

0.259 0.006
0.298 0.022
0.687 0.073 a
(0 3 ; 0 = 6 )

0.259 0.006
0.307 0.022
0.410 0.077 ab
(0 = 3 ; 0 = 6 )

0.259 0.006
0.333 0.024
0.378 0.074 b
(0 = 3 )

0.259 0.006
0.281 0.026
0.319 0.057 b
ns

FDI
Baseline
3 months
6 months
t-Testa

0.629 0.085
1.075 0.180
6.418 0.823 a
(0 = 6 )

0.629 0.085
1.627 0.437
2.921 0.516 b
(0 = 6 )

0.629 0.085
1.237 0.124
1.442 0.249 b
(0 = 3 ; 0 = 6 )

0.629 0.085
0.991 0.184
1.363 0.349 b
ns

ns

ns

ns

ns

ns, not significant; significant, P < 0.05; significant, P < 0.01; significant, P < 0.001.
a
t-Test between Baseline (0) and exposure time; b ANOVA between areas. For each metal the
values in the rows with the same letter do not differ significantly, P < 0.05 (Tukey pairwise
comparison of means).

TILLANDSIA AS AIR POLLUTION BIOMONITORS

207

the rates of metal accumulation (g. g1 . month1 ) were higher in this period as
well as the associated physiological responses.
It is important to note that the concentrations of gaseous contaminants (SO2 ,
NOx , etc,), as well as those of particulate matter, are reduced in the citys air during
the spring and summer months due to the increased rainfall, a situation that was
evidenced by the higher rates of deposition. This was reflected in the physiological
response of all the studied biomonitors that had lower values of membrane lipid
peroxidation products (HPCD and MDA) and lower chlorophyll a degradation
values during the second period, except for the industrial area. Nevertheless, after
3 months exposure for T. tricholepis and 6 months exposure for T. capillaries, the
values of the FDI allowed to distinguish the more contaminated sites (Southeast
and Centre) from the less contaminated areas (North and Southwest).

4. Conclusions
Of the four species studied, T. capillaris was the one with the greatest correspondence between metal accumulation (except for Zn) and total atmospheric deposition
levels. Therefore, we can suggest its use as a biomonitor for the accumulation of
Fe, Mn, Cu, Co, Ni and Pb.
The metals with the highest levels of enrichment in the biomonitors were Cu,
Zn and Pb, elements clearly related to anthropogenic emission sources (urban and
industrial) as also indicated by their concentrations in each area. The enrichment
pattern of the concentration of Zn in T. capillaris, T. permutata and T. retorta was
not coincident with the concentration of this element in the deposition samples.
However, the accumulation of Zn in T. tricholepis did correlate with the values of
this metal in the deposition samples. The highest enrichment levels of the analysed
metals were found in T. capillaris followed by T. tricholepis, T. permutata and
T. retorta.
The calibration of these four species of Tillandsia as physiological response
biomonitors indicated that even though damage (measured with the FDI) was observed in one of the species after 3 months exposure (T. tricholepis), in general this
exposure time was not enough. Therefore, for T. capillaris, T. permutata and T. retorta clear differences of air pollution levels between the areas using the variation
of physiological parameters could only be established after 6 months exposure.
The FDI proved to be a good biomarker of the effect of pollutants on T. capillaris, making it an interesting tool to evaluate atmospheric quality when using this
biomonitor. As previously shown for the physiological response biomonitors, the
order of efficiency proposed for these species is: T. tricholepis > T. capillaris > T.
retorta > T. permutata.
Finally, although not the object of the current investigation, it is important to
note the elevated levels of heavy metals (Cu, Ni, Fe, Co, Mn and Pb) found in the
samples of total atmospheric deposition in an industrialized area inside the city of

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E. D. WANNAZ AND M. L. PIGNATA

Cordoba. Furthermore, elevated levels of Zn were found in the centre of the city
and were even higher than those of the industrialized area. The presence of this
metal downtown is mainly associated with vehicular traffic and considering the
high population density of the area these elevated levels of Zn are hazardous for
physiological response biomonitors the populations health.
Acknowledgements
This work was supported partially by the Agencia Nacional de Promocion Cientifica
y Tecnica (FONCyT), the Brazilian Synchrotron Light Laboratory (LNLS) and
Secretaria de Ciencia y Tecnica de la Universidad Nacional de Cordoba (SECyTUNC). We are especially grateful to Dr. Carlos A. Perez for his technical assistance
in the measurement of metals with TXRF.
References
Calasans, C. F. and Malm, O., 1997: Elemental mercury contamination survey in a chlor-alkali plant
by the use of transplanted Spanish moss, Tillandsia usneoides (L.), Sci. Total Environ. 208,
165177.
Caussy, D., Gochfeld, M., Gurzau, E., Neagu, C. and Ruedel H., 2003: Lessons from case studies of
metals investigating exposure, bioavailability and risk, Ecotox. Environ. Safety. 25, 4551.
Chaphebar, S. B., 2000: Phytomonitoring in Industrial Areas, in S. B. Agrawal and M. Agrawal (eds),
Environmental Pollution and Plant Responses, Lewis Publishers, New York, pp. 329342.
Cuny, D., Pignata, M. L., Kranner, I. and Beckett, R., 2002: Biomarkers of pollution-induced oxidative stress and membrane damage in lichens, in P. Nimis (ed.), Section 1: Lichens as Indicators of Pollution, Lichen Monitoring, Kluwer Academic Publishers, The Netherlands, pp:
97111.
Cuny, D., Van Haluwyn, C. and Shirali, P., 2000: The concept of biomarker in lichenology - Application
in bioindication survey, in Progress and Problems in Lichenology at the turn of the millennium,
Reports of the Fourth IAL Symposium, Barcelona.
Figueiredo, A. M. G., Saiki, M., Ticianelli, R. B., Domingos, M., Alves, E. S. and Market, B.,
2001: Determination of trace elements in Tillandsia usneoides by neutron activation analysis for
environmental biomonitoring, J. Radioanal. Nucl. Ch. 249, 391395.
Flores, F. E. V., 1987: O uso de plantas como bioindicadores de puluicao no ambiente urbanoindustrial: experiencias en Porto Alegre, RS Brasil, Tubinger. Geogr. Studien. 96, 7986.
Gonzalez, C. M. and Pignata, M. L., 1994: The influence of air pollution on soluble proteins, chlorophyll degradation, MDA, sulphur and heavy metals in a transplanted lichen, Chem. Ecol. 9,
105113.
Harrison, R. M. and Johnston W. R., 1985: Deposition fluxes of lead, cadmiun, copper and polynuclear aromatic hydrocarbons (PAH) on the verges of a major highway, Sci. Total Envriron. 46,
121135.
Hewitt, C. N. and Rashed, M. B., 1991: The deposition of selected pollutants adjacent to a major rural
highway, Atmos. Environ. 25, 979983.
Kosugi, H., Jojima, T. and Kikugawa, K., 1989: Thiobarbiturir acid-reactive substances from peroxidized lipids, Lipids. 24, 873881.
Levin, A. G. and Pignata, M. L., 1995: Ramalina ecklonii (Spreng.) Mey. and Flot, as a bioindicator
of atmospheric pollution in Argentina, Can. J. Bot. 73, 11961202.

TILLANDSIA AS AIR POLLUTION BIOMONITORS

209

Malm, O., De Freitas Fonseca, M., Hissnauer Miguel, P., Rodriguez Bastos, W. and Neves Pinto, F.,
1998: Use of epiphyte plants as biomonitors to map atmospheric mercury in a gold trade centre
city, Amazon, Brazil. Sci. Total Environ. 213, 5764.
Manta, D. S., Angelone, M., Bellanca, A., Neri, R., Sprovieri, M., 2002: Heavy metals in urban soils:
a case study from the city of Palermo (Sicily), Italy, Sci. Total Environ. 300, 229243.
Olcese, L. E. and Toselli, B. M., 2002: Some aspects of air pollution in Cordoba, Argentina. Atmos.
Environ. 36, 299306.
Pignata, M. L., Gudino, G. L., Wannaz, E. D., Pla, R. R., Gonzalez, C. M., Carreras, H. A. and
Orellana, L., 2002: Atmospheric quality and distribution of heavy metals in Argentina employing
Tillandsia capillaris as a biomonitor, Environ. Pollut. 120, 5968.
Schrimpff, E., 1981: Air pollution patters in two cities in Colombia S.A. according to trace substance
content of an epiphytic (Tillandsia recurvata L.), Water Air Soil Poll. 21, 279315.
Stein, A. F. and Toselli, B. M., 1996: Street level air pollution in Cordoba City, Argentina, Atmos.
Environ. 30, 34913495.
Tiller, K. G., 1992: Urban soil contamination of soils in urban areas, Aust. J. Soil Res. 30, 937957.
Viard, B., Pihan, F., Promeyrat, S. and Pihan, J. C., 2004: Integrated assessment of heavy metal (Pb,
Zn, Cd) highway pollution: bioaccumulation in soil, Graminaceae and land snails, Chemosphere.
55, 13491359.
Wannaz, E. D., Zygadlo, J. A. and Pignata, M. A., 2003: Air pollutants effect on monoterpenes
composition and foliar chemical parameters in Schinus areira L, The Sci. of Total Environ. 305,
177193.