Calibration of Four Species of Tillandsia as Air

Journal of Atmospheric Chemistry (2006) 53: 185209DOI: 10.10007/s10874-005-9006-6 C Springer 2006

Calibration of Four Species of Tillandsia as AirPollution Biomonitors

EDUARDO D. WANNAZ and MARIA L. PIGNATAInstituto Multidisciplinario de Biologia Vegetal (IMBIV), Consejo Nacional de InvestigacionesCientificas y Tecnicas (CONICET). Catedra de Quimica General. Facultad de Ciencias Exactas,Fisicas y Naturales. Universidad Nacional de Cordoba. Avda. Velez Sarsfield 1611. CiudadUniversitaria (X5016 GCA) Cordoba, Argentina, e-mail: [email protected]@com.uncor.edu.

(Received: 3 January 2005; accepted: 17 September 2005)Abstract. Many organisms have been used as bioindicators of atmospheric contamination, withmoss and lichen species being the most common. However, studies using epiphytic vascular speciesof Tillandsia have shown a good correlation between the presence of pollutants and the bioindicatorsresponse. Therefore, the aim of our investigation was to calibrate and compare the response of fourTillandsia species of Argentina to ascertain whether they could be used as atmospheric contaminationbiomonitors. For this, we analysed the correlation between the levels of heavy metals in total atmo-spheric deposition samples and: a) their rate of enrichment; b) the physiological response of the plantsamples.

Tillandsia samples collected from a non contaminated area in the province of Cordoba were trans-planted to four areas in the capital city with different sources of pollution (industrial or traffic emis-sions). 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. Simultaneouslysamples 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 auseful tool to evaluate different levels of atmospheric quality in these species. The rate of heavy metaldeposition was higher in the industrial area for all metals except for Zn whose values were higher inareas 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 ofenvironmental quality because it provides the integrated effect of all the environ-mental factors including air pollution and weather conditions. Furthermore, whenbiomonitoring is combined with physiochemical monitoring, the relationship be-tween 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 airpollution biomonitors as they are not in contact with the soil, as their source of

186 E. D. WANNAZ AND M. L. PIGNATA

water and nutrients is atmospheric. For this reason, their elemental tissue contentlargely reflects the atmospheric levels of some toxic elements (Figueiredo et al.,2001). T. usneoides has proven to efficiently accumulate atmospheric Hg presentin the neighbouring area of a chlor-alkali plant in Rio de Janeiro (Brazil) (Calasansand Malm, 1997), as well as in urban areas (Malm et al., 1998). T. aeranthos andT. recurvata have been used in Porto Alegre (Brazil) to determine the atmosphericcontent of sulphur and heavy metals in both industrialized and residential areas(Flores, 1987). In Colombia, the airborne heavy metal deposition in the highlyindustrialized Cauca Valley was studied using T. recurvata as an accumulativeindicator (Schrimpf, 1984).

However, in order to establish and use epiphytic plants as air quality biomonitorsit is important to characterize the response of each species to different environmentalconditions, as well as to different levels of pollutants. Many species of Tillandsiagrow in a large area of Argentina; among them, T. capillaris has been used in apreliminary study as a passive biomonitor of atmospheric contamination (Pignataet al., 2002). We therefore considered that T. capillaris, T. permutata, T. tricholepisand T. retorta, species that are abundant in the province of Cordoba, could be usedto detect the emission sources of heavy metals and to establish different levels ofair quality via active biomonitoring studies.

The aims of the present investigation were to: (a) calibrate the heavy metal ac-cumulation 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 eachspecies 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 ofthe Republic of Argentina (3124 S, 6411 W) at an altitude of approximately400 m above sea level (Figure 1). It has a population of around 1.5 million andan irregular topography. Its general structure is funnel-shaped, with an increasingpositive slope from the centre towards the surrounding areas. This somewhat con-cave formation reduces air circulation and causes frequent thermal inversions inboth autumn and winter. The climate is subhumid, with an average annual rainfallof 790 mm, concentrated mainly in summer. The mean annual temperature is of17.4C and the prevailing winds come from the NE, S and SE.

The main source of air pollution in Cordoba city (downtown) is from mobilesources (Stein and Toselli, 1996). The traffic pattern is closely related to all the pri-mary pollutants measured (CO, NOx and PM10) which is another reason further jus-tifying the assumption that the citys pollution is mainly due to mobile sources (Ol-cese and Toselli, 2002). Cordoba city also has an important industrial developmentof 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 biomonitorswere transplanted and where the samples of total atmospheric deposition were taken().

2.2. BIOLOGICAL MATERIAL, PLANT TRANSPLANTATION AND TOTALDEPOSITION SAMPLES

Tillandsia capillaris Ruz & Pav. f. capillaris, Tillandsia permutata A. Cast.,Tillandsia tricholepis Baker and Tillandsia retorta Griseb. ex Baker, were col-lected from the Dique la Quebrada, a natural reserve considered an unpollutedsite found in the north of the province of Cordoba at approximately 40 km from thecapital city.

Plant bags containing 200 g plant material were prepared according to Gonzalezand Pignata (1994) and transplanted simultaneously to four areas in the city ofCordoba with different conditions of atmospheric contamination. In each area sixbags were bags were hung 3 m above ground level (n = 6 bags/species/site). Theprocess of transplantation was held on the 14th of June 2002 and corresponds tothe winter season in which the lowest rainfall values and temperatures are recorded


Table I. Mean values of rainfall and temperature in thecity of Cordoba during the months of June to December2002

Month Rainfall (mm) T (Min-Max) (C)

June 0.0 8.4 (1.1 15.7)July 26.5 9.2 (2.2 16.2)August 23.0 11.7 (3.9 19.6)September 25.5 13.7 (5.2 22.2)October 32.0 18.9 (11.3 26.5)November 259.5 21.6 (14.4 29.9)December 117.0 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 Tillandsiasamples were conserved at 15C in the dark until the corresponding chemicalanalyses were undertaken. Three assays were taken for each sample in every deter-mination.

In order to establish the initial state of the samples before transplantation abasal sample from the original collection site was analysed for each species. Thesesamples were used as the controls to which the transplanted samples were comparedto evaluate the changes in metal concentration and the physiological responses ofeach species exposed to atmospheric contamination.

For the evaluation of the deposit flux, suitable experimental devices were adaptedfrom those described previously (Harrison and Johnston, 1985; Hewitt and Rashed,1991) to collect wet and dry deposition. The device consisted of a polypropy-lene bottle with a funnel (20 cm diameter) that was located 3 m above groundlevel in order to avoid soil interferences and was covered with a PVC laminatednet to prevent the entrance of any undesired objects (insects, leaves). The deviceswere hung on the same day the plant material was transplanted. Half the deviceswere 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 be-ing 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). Twoof these sites corresponded to highly contaminated places, one by traffic (Centre)and the other by industrial activities (Southeast). The other two corresponded toresidential areas where high levels of atmospheric contamination are not expected.


Southeast area: an industrial area, with metallurgic and metal-mechanicindustries. The sampling site was located inside the premises of an industrial plantin which metallic parts are made (tanks, radiators, covers, etc).

Centre area: the site was located downtown in a densely populated area. It iswhere most of the public buildings, government offices, shops and businesses arelocated and almost all the local buses run through it. The centre of the city is foundin a topographic depression and therefore the dispersion of the contaminants isdifficult.

North area: a suburban residential area in which houses and small and mediumshops are found. Traffic is much less than the centre area and is mainly composedof cars with some public transport in the main avenues.

Southwest area: also a residential area with similar characteristics to the Northsite, 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 ofTillandsia (3g dry weight) taken from the plant samples that had been transplantedfor 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 500C for4 hours. The ashes were digested with HCl (18%):HNO3 (3:1), the solid residuewas separated by centrifugation and the volume was adjusted to 25 mL with Milli-Qwater. After this, 10 ppm of a Ge solution was added as an internal standard. Aliquotsof 5 L were taken from this solution and dried on an acrylic support. As a qualitycontrol, blanks and samples of the standard reference material Hay IAEA-V-10were prepared in the same way. Standard solutions with known concentrations ofdifferent elements and Ge as an internal standard were prepared for the calibrationof the system.

The samples were measured for 200 s, using the total reflection set up mountedat the X-ray fluorescence beamline of the National Synchrotron Light Laboratory(LNLS), Campinas, Brazil. For the excitation, a polychromatic beam was usedof 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 6months after exposure) were evaporated until dry and the residue was heated inan oven at 500C for 4 hours. The ashes were treated in the same way as those ofthe plant samples in order to prepare the metallic acid solutions to be analysed byTRXF. Results are expressed as g/m2.exposure time.


2.6. DRY WEIGHT/FRESH WEIGHT RATIO

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

2.7. CHLOROPHYLL CONTENT

Chlorophyll extracts were prepared from 200 mg of plant material that was ho-mogenised in 10 mL EtOH 96 % V/V with an Ultra Turrax homogeniser and thesupernatant was then separated by centrifugation. In order to measure phaeophytinformation, 0.06 M HCl was added to clear the chlorophyll extract (1 mL HCl and 5mL chlorophyll extract). Absorption of chlorophylls and phaeophytins, and that ofphaeophytins alone (after HCl addition) was measured with a Beckman DU 7000spectrophotometer. Concentrations of chlorophylls and phaeophytins were calcu-lated on a fresh weight basis (Pignata et al., 2002). The chlorophyll b/ chlorophylla (Chl-b /Chl-a) ratios were also calculated. The results are expressed in mg.g1FW.

2.8. SULPHUR CONTENT

Five millilitres of a Mg (NO3)2 saturated solution were added to 0.5 g plant materialand dried in an electric heater. Subsequently, the sample was heated in an oven for 30min at 500C. The ashes were then suspended in 6 M HCl, filtered and the resultingsolution was boiled for 3 minutes. Finally the solution was brought to 50 mLwith distilled water.

The amount of SO24 in the solution was determined by the acidic suspensionmethod with BaCl2 which subsequently allowed to calculate the sulphur contentof each sample (Gonzalez and Pignata, 1994). The concentration was expressed inmg of total sulphur.g1 FW.

2.9. ESTIMATION OF PEROXIDATION PRODUCTS

The malondialdehyde (MDA) content was measured by a colorimetric method. Theamount 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 ofthe plant material in 96% V/V EtOH at a ratio of 1:50 FW/V. The absorption wasmeasured in the supernatant at 234 nm and its concentration was calculated using = 2.65 104 M1cm1 (Levin and Pignata, 1995). The results were expressedas mol g1 FW.


2.10. FOLIAR DAMAGE INDEX

A Foliar Damage Index (FDI) was calculated in order to combine the changes ofeach 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 chorophylla concentration in mg g1 FW; S is the sulphur content in mg 1 FW; MDA is themalondialdehyde concentration in nmol g1 FW; HPCD is the hydroperoxy conju-gated dienes in mol g1 FW. The parameter with subindex b in the denominatorrepresents the arithmetic mean values calculated in the basal samples.

The FDI was used to compare the physiological response of the four studiedspecies to the presence of air pollutants and therefore the values were related to thedifferent levels of air quality.

2.11. STATISTICAL ANALYSIS

Results are expressed as the mean values of three independent determinations foreach species in each sampling area. The data was analysed using a paired t-Test ofANOVA 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 samplesfor each sampling area after 3 and 6 months exposure. For both periods, the con-centration 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. Inthe samples corresponding to the 6 month period the concentrations of Co weresignificantly higher in the Centre and Southeast areas than the North and Southwestareas. Therefore, highest levels of metal deposition in the industrial area correspondto the emissions of the metallurgic and metal-mechanic industries present in thearea.

On the other hand, the highest levels of Zn deposition were related to trafficemission as they corresponded to the Centre area. High levels of Zn have beenreported for other areas in which the dispersion of traffic emission contaminants isinfluenced by topographical characteristics and meteorological conditions (Viardel at., 2004). Due to the fact that traffic pollutants include toxic metals such aslead, cadmium and zinc (Caussy et al., 2003), these elevated levels of Zn couldhave adverse effects on the populations health. After 6 months deposition elevated


Table II. Mean values (S.D.) of the amount of metal found in total atmospheric depositionsamples after 3 and 6 months transplantation, including the results of the ANOVA amongdifferent areas

Deposition (g/m2 after 3 months)Centre Southeast North SouthwestMean S.D. Mean S.D. Mean S.D. Mean S.D. ANOVA

Mn 6.09 1.13 b 18.00 2.51 a 4.29 1.53 b 7.48 1.46 b Fe 409.58 66.60 b 1573.20 528.96 a 236.08 117.22 b 422.13 118.30 b Co 18.42 3.35 ab 27.72 9.20 a 3.85 1.31 b 6.00 1.02 b Ni 3.49 0.34 b 7.72 0.23 a 2.92 0.33 b 3.58 0.87 b Cu 9.75 0.84 b 29.71 1.11 a 1.96 0.86 c 2.99 1.34 c Zn 797.33 41.29 a 28.69 0.35 b 12.39 2.70 b 10.77 2.99 b Pb 6.60 1.55 b 18.35 4.58 a 2.76 0.52 b 2.17 0.50 b

Deposition (g/m2 after 6 months)Mn 8.14 0.37 b 31.40 3.85 a 11.54 2.65 b 19.98 4.19 ab Fe 647.84 40.75 b 2762.84 259.61 a 536.83 293.43 b 688.97 194.21 b Co 50.82 2.35 a 54.98 2.61 a 11.03 1.65 b 12.01 1.93 b Ni 10.26 0.60 ab 14.67 1.95 a 4.24 1.67 c 5.90 0.18 bc Cu 25.40 1.77 b 59.06 10.51 a 2.96 0.57 c 4.53 0.06 bc Zn 3965.01 643.38 a 98.98 35.44 b 42.42 11.64 b 20.51 1.11 b Pb 14.43 1.36 b 34.08 7.31 a 6.84 1.98 b 3.71 1.06 b

ns. not significant; significant with P < 0.05; significant with P < 0.01; significant withP < 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 theseresults, and as these are the first for Cordoba city regarding the emission of heavymetals, we believe that further research is necessary to determine their atmosphericlevels as there is currently no organism monitoring the atmospheric contaminationof this city.

Viard et al. (2004) have informed that in an area near a highway in France theamount of metal deposition was in decreasing order [Zn] > [Pb] > [Cd] in gm2 d1. It is therefore possible that these metals are enriched in urban soils andso become dispersed as particulate atmospheric matter downtown. The elevatedlevels 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 exposedpopulations health. Urban soils are known to have peculiar characteristics suchas poor structure and high concentrations of trace elements (Tiller, 1992). Thus,they are recipients of large amounts of heavy metals from a variety of sourcesincluding vehicle emissions, cool burning wastes and other activities (Manta et al.,2002).


3.2. 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 eacharea (Centre, Southeast, Southwest and North) are shown in Tables III, IV, V andVI respectively.

In T. capillaris (Table III) the concentration of all metals increased after 3and 6 months of exposure in all the study areas, with maximal enrichment valuesfor the Southeast area (industrial). This was observed even for Zn, although thismetal had the greatest deposition rates in the Centre area. The lowest values ofmetal concentration corresponded to the samples transplanted to the residentialareas (North and Southwest) in accordance with the values of total atmosphericdeposition. After 3 months exposure some metals showed significant differencescompared to the basal samples, although there were no differences between thesampling areas. We can therefore conclude that 3 months is not an adequate timeof 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 studyareas after 6 months exposure. No significant differences were found for Mn withrespect to the basal values for both exposure periods. On the other hand, the highestconcentrations 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 inT. tricholepis (Table V) compared to the basal samples, this species only allowed todifference the levels of Cu, Zn and Pb between the sampling areas, with the highestlevels 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 be-ing the site with the highest accumulation of Cu and the Southwest area the onewith the highest accumulation of Zn. After 6 months transplantation, all the metalconcentrations were increased with respect to the basal values. Thus, the highestconcentrations 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 andSouthwest sites.

Regarding the metal content of the biomonitors, the only two species that alloweddifferencing the level of Mn accumulation between the study areas, and only after6 months exposure, were T. capillaris and T. retorta. The Fe content in the trans-planted samples increased in all the species and in almost all the areas compared tothe basal samples. However, the accumulation of this metal was only significant be-tween the areas for T. capillaris, T. permutata y T. retorta and reflected the pattern ofdeposition measured for this element. The pattern of deposition for Co was similarto that of Fe and Mn and T. capillaris and T. retorta were the species that allowedto discriminate the variation of this metal between the sampling areas. Total Co


Table III. Comparison of the mean values of heavy metal concentrations (S.D.) measuredin T. capillaris. The values represent the result of a t-Test between the baseline sample andthe transplanted samples (3 and 6 months exposure) and the ANOVA between the differenttransplantation areas

Mean SDCentre Southeast North Southwest ANOVAb

MnBaseline 13.579 0.277 13.579 0.277 13.579 0.277 13.579 0.2773 months 24.872 3.016 16.187 4.663 21.616 2.492 26.894 2.288 ns6 months 58.107 5.349 b 85.052 5.290 a 45.393 1.567 b 24.881 3.376 c t-Testa (0 = 3; 0 = 6) (0 = 6) (0 = 3; 0 = 6) (0 = 3; 0 = 6)FeBaseline 197.04 38.63 197.04 38.63 197.04 38.63 197.04 38.633 months 291.14 47.71 269.17 75.67 312.76 13.31 287.93 34.723 ns6 months 1001.62 100.83 b 1933.67 143.24 a 879.52 139.59 b 503.97 27.86 c t-Testa (0 = 6) (0 = 6) (0 = 3; 0 = 6) (0 = 6)CoBaseline 0.087 0.035 0.087 0.035 0.087 0.035 0.087 0.0353 months 0.133 0.023 0.117 0.030 0.119 0.002 0.120 0.001 ns6 months 0.382 0.051 b 0.725 0.089 a 0.291 0.029 bc 0.193 0.014 c t-Testa (0 = 6) (0 = 6) (0 = 6) (0 = 6)NiBaseline 0.220 0.024 0.220 0.024 0.220 0.024 0.220 0.0243 months 0.257 0.001 0.362 0.095 0.347 0.017 0.348 0.069 ns6 months 0.927 0.034 b 1.796 0.132 a 0.660 0.082 bc 0.416 0.081 c t-Testa (0 = 6) (0 = 6) (0 = 3; 0 = 6) (0 = 6)CuBaseline 1.249 0.142 1.249 0.142 1.249 0.142 1.249 0.1423 months 1.591 0.151 2.883 0.454 1.345 0.188 2.260 0.618 ns6 months 5.007 0.075 b 32.696 2.432 a 3.841 0.533 b 2.051 0.182 b t-Testa (0 = 6) (0 = 3; 0 = 6) (0 = 6) (0 = 6)ZnBaseline 4.003 0.126 4.003 0.126 4.003 0.126 4.003 0.1263 months 7.326 1.306 7.259 2.822 6.024 1.430 6.883 0.472 ns6 months 19.806 0.815 b 47.248 4.447 a 15.882 3.421 b 6.551 0.594 c t-Testa (0 = 3; 0 = 6) (0 = 6) (0 = 6) (0 = 3; 0 = 6)PbBaseline 0.675 0.196 0.675 0.196 0.675 0.196 0.675 0.1963 months 1.153 0.190 1.244 0.092 0.613 0.291 0.515 0.170 ns6 months 4.870 0.426 b 13.945 0.231 a 3.675 0.251 b 1.405 0.476 c t-Testa (0 = 6) (0 = 3; 0 = 6) (0 = 6) ns

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


Table IV. Comparison of the mean values of heavy metal concentration (SD) measured inT. permutata. The values represent the result of a t-Test between the baseline sample andthe transplanted samples (3 and 6 months exposure) and the ANOVA between the differenttransplantation areas


MnBaseline 20.927 1.946 20.927 1.946 20.927 1.946 20.927 1.9463 months 20.111 1.188 21.812 1.635 13.791 8.083 21.341 1.383 ns6 months 30.559 10.513 54.945 14.875 45.767 14.094 35.449 5.331 nst-Testa ns ns ns ns

FeBaseline 279.34 25.65 279.34 25.65 279.34 25.65 279.34 25.653 months 314.22 54.63 308.18 4.02 211.126 76.29 271.41 13.63 ns6 months 530.11 140.86 1364.79 330.62 799.38 246.52 679.77 93.31 nst-Testa ns (0 = 6) ns (0= 6)CoBaseline 0.115 0.004 0.115 0.004 0.115 0.004 0.115 0.0043 months 0.137 0.022 0.130 0.014 0.100 0.044 0.117 0.004 ns6 months 0.202 0.064 b 0.485 0.103 a 0.309 0.007 ab 0.213 0.017 b t-Testa ns (0 = 6) (0 = 6) (0 = 6)NiBaseline 0.379 0.023 0.379 0.023 0.379 0.023 0.379 0.0233 months 0.397 0.060 0.498 0.067 0.315 0.186 0.407 0.096 ns6 months 0.593 0.091 1.392 0.616 0.766 0.007 0.595 0.100 nst-Testa ns ns (0 = 6) nsCuBaseline 1.913 0.285 1.913 0.285 1.913 0.285 1.913 0.2853 months 2.047 0.093 ab 3.410 0.611 a 1.354 0.571 b 2.105 0.589 ab 6 months 4.112 1.572 b 19.527 3.646 a 3.433 0.831 b 3.487 0.527 b t-Testa ns (0 = 6) ns nsZnBaseline 12.597 0.251 12.597 0.251 12.597 0.251 12.597 0.2513 months 30.072 1.182 a 22.991 6.086 ab 20.117 4.307 ab 12.693 1.844 b 6 months 41.779 8.255 ab 42.612 9.006 ab 65.649 8.453 a 17.851 1.100 b t-Testa (0 3; 0 = 6) (0 = 6) (0 = 6) (0 = 6)PbBaseline 0.812 0.112 0.812 0.112 0.812 0.112 0.812 0.1123 months 0.473 0.117 0.285 0.114 0.731 0.268 0.367 0.056 ns6 months 1.741 0.346 b 11.216 1.662 a 3.292 0.447 b 3.398 1.511 b t-Testa ns (0 = 3; 0 = 6) (0 = 6) (0 = 3)

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


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


MnBaseline 16.192 1.345 16.192 1.345 16.192 1.345 16.192 1.3453 months 26.529 0.437 27.455 1.024 26.298 5.008 18.298 0.923 ns6 months 45.175 1.570 58.932 24.38 78.543 5.995 46.291 16.265 nst-Testa (0 = 3; 0 = 6) (0 = 3) (0 = 6) nsFeBaseline 184.77 34.71 184.77 34.71 184.77 34.71 184.77 34.713 months 293.83 14.04 344.53 56.47 357.39 50.67 209.08 3.09 ns6 months 951.60 111.15 1336.01 277.68 1090.51 113.48 819.65 391.40 nst-Testa (0 = 6) (0 = 6) (0 = 6) nsCoBaseline 0.102 0.015 0.102 0.015 0.102 0.015 0.102 0.0153 months 0.164 0.017 0.139 0.033 0.168 0.025 0.096 0.028 ns6 months 0.308 0.010 0.546 0.078 0.376 0.058 0.275 0.119 nst-Testa (0 = 6) (0 = 6) (0 = 6) nsNiBaseline 0.248 0.042 0.248 0.042 0.248 0.042 0.248 0.0423 months 0.197 0.121 0.234 0.048 0.261 0.037 0.209 0.026 ns6 months 0.706 0.157 1.080 0.140 0.718 0.200 0.467 0.054 nst-Testa ns (0 = 6) ns (0 = 6)CuBaseline 1.095 0.217 1.095 0.217 1.095 0.217 1.095 0.2173 months 1.526 0.312 2.835 0.729 3.464 2.006 0.931 0.272 ns6 months 7.239 1.532 b 21.425 2.312 a 3.863 0.058 b 2.600 0.316 b t-Testa (0 = 6) (0 = 6) (0 = 6) (0 = 6)ZnBaseline 3.128 1.175 3.128 1.175 3.128 1.175 3.128 1.1753 months 4.692 0.512 5.339 1.641 5.868 1.369 4.077 0.064 ns6 months 24.906 3.924 a 20.723 4.797 ab 10.891 1.575 b 7.532 1.797 b t-Testa (0 = 6) (0 = 6) (0 = 6) nsPbBaseline 0.318 0.124 0.318 0.124 0.318 0.124 0.318 0.1243 months 0.510 0.089 0.769 0.169 0.547 0.265 0.373 0.102 ns6 months 1.701 0.010 ab 3.858 1.071 a 1.748 0.396 ab 0.472 0.006 b t-Testa (0 = 6) (0 = 6) (0 = 6) ns

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


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


MnBaseline 17.468 2.655 17.468 2.655 17.468 2.655 17.468 2.6553 months 15.673 4.747 19.509 2.489 26.781 1.735 17.958 0.281 ns6 months 21.326 0.594 bc 38.014 5.869 a 31.841 0.578 ab 12.854 2.214 c t-Testa ns (0 = 6) (0 = 3; 0 = 6) nsFeBaseline 155.01 12.56 155.01 12.56 155.01 12.56 155.01 12.563 months 138.95 51.15 239.32 19.24 170.99 39.97 169.86 19.62 ns6 months 288.64 71.61 b 857.99 122.43 a 442.178 76.88 b 152.24 34.56 b t-Testa (0 = 6) (0 = 3; 0 = 6) (0 = 6) nsCoBaseline 0.057 0.009 0.057 0.009 0.057 0.009 0.057 0.0093 months 0.120 0.038 0.092 0.015 0.113 0.048 0.071 0.027 ns6 months 0.105 0.023 b 0.301 0.040 a 0.134 0.011 b 0.071 0.016 b t-Testa (0 = 6) (0 = 3; 0 = 6) (0 = 6) nsNiBaseline 0.454 0.024 0.454 0.024 0.454 0.024 0.454 0.0243 months 0.402 0.083 0.430 0.074 0.565 0.136 0.367 0.018 ns6 months 0.574 0.068 1.005 0.118 0.882 0.176 0.667 0.208 nst-Testa ns (0 = 6) (0 = 6) (0 = 3)CuBaseline 1.397 0.108 1.397 0.108 1.397 0.108 1.397 0.1083 months 2.043 0.342 ab 3.234 0.307 a 2.462 0.466 ab 1.535 0.060 b 6 months 2.184 0.105 b 8.213 0.199 a 2.051 0.171 b 1.321 0.174 c t-Testa (0 = 3; 0 = 6) (0 = 3; 0 = 6) (0 = 3; 0 = 6) nsZnBaseline 8.114 4.533 8.114 4.533 8.114 4.533 8.114 4.5333 months 4.009 0.840 b 5.635 1.067 b 6.192 0.025 b 35.315 0.172 a 6 months 39.434 3.487 a 13.264 0.965 c 23.705 4.028 bc 34.259 3.470 ab t-Testa (0 = 6) ns (0 = 6) (0 = 3; 0 = 6)PbBaseline 0.363 0.127 0.363 0.127 0.363 0.127 0.363 0.1273 months 0.409 0.060 0.703 0.099 0.440 0.152 0.369 0.136 ns6 months 0.695 0.102 ab 1.656 0.297 a 0.620 0.398 b 0.538 0.021 b t-Testa ns (0 = 6) ns ns

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


deposition was greater in the Southeast and Centre areas and this was reflected in theT. capillaris samples that were the ones that accumulated the highest concentrationsof this element.

Concerning Ni, T. capillaris was able to distinguish areas with different levelsof accumulation after 6 months exposure with a pattern that coincided with the totaldeposition values measured.

The enrichment levels of Cu found in T. permutata and T. retorta differencedthe industrial area from the rest of the sites after only 3 months exposure, whereasthe rest of the species manifested this difference only after being transplanted for6 months.

The levels of the concentration of Zn in samples transplanted for 6 months weresignificantly higher than the basal samples for all species. However, whereas T.capillaris showed differences between areas with the highest values belonging tothe Southeast site, in T. permutata the highest values corresponded to the North,Centre and Southeast areas and T. tricholepis and T. retorta showed the highestvalues for the Centre and Southeast areas. It is important to note that these twolast species showed a pattern of Zn accumulation coincident with the levels of totaldeposition.

With respect to Pb, T. capillaris and T. permutata showed a similar behaviourhaving the highest levels of enrichment in the Southeast (industrial) area in samplesexposed for 6 months. T. retorta had the highest concentrations of this metal in theSoutheast and Centre areas and T. tricholepis in the Southeast, Centre and Northsites. It is interesting to note that in all the species the highest concentrations of Pbcorresponded 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, theenrichment factors for each metal in every species were calculated according to thefollowing equation:

EF = Xi j/Xb

where Xi j is the concentration of element X in mg g1 FW in time i and sitej; Xb is the concentration of element X in mg g1 FW in the basal samples. Themaximal values of enrichment for each metal in each of the four species are shown inTable VII.

Cu, Zn and Pb were the metals most enriched in T. capillaris, T. permutataand T. tricholepis indicating their anthropogenic origin. Among these species,T. capillaris had the highest accumulation of all the metals analysed thus exhibitinga greater bioaccumulation capacity. On the contrary, T. retorta was the species thatshowed the lowest levels of enrichment for all metals.


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

T. capillaris T. permiutata T. tricholepis T. retorta

Mn 6.26 2.63 4.85 2.18Fe 9.81 4.86 7.23 5.54Co 8.33 4.22 5.35 5.28Ni 8.16 3.67 4.35 2.21Cu 26.18 10.21 19.57 5.88Zn 11.80 5.21 7.96 4.86Pb 20.66 13.81 12.13 4.56

Table VIII. Lineal regression coefficients (R2) 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 TotalDeposition

T. capillaris T. permutala T. tricholepis T. retorta

R2 a R2 a R2 a R2 a

Mn 0.27 1.33 0.50 1.21 0.17 0.98 0.16 0.39Fe 0.55 0.51 0.53 0.35 0.27 0.27 0.66 0.24Co 0.64 0.009 0.38 0.004 0.38 0.005 0.36 0.002Ni 0.79 0.11 0.55 0.07 0.58 0.06 0.27 0.03Cu 0.76 0.46 0.63 0.26 0.76 0.29 0.82 0.10Zn 0.02 0.001 0.06 0.003 0.45 0.004 0.19 0.004Pb 0.77 0.37 0.57 0.26 0.70 0.09 0.79 0.04

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

3.4. LINEAL REGRESSION

In order to compare the ability to accumulate atmospheric metals of the four Tilland-sia species, lineal regressions were made between the metal content of the totaldeposition 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. Parametera, that represents the increase of the concentration of a determined metal in thebiomonitor that arises with the increase of one unit in the total deposition valuesof the metal, was very low for Co and Zn in all the studied species indicating theirvery low efficiency to accumulate these elements. On the other hand, the differencesbetween the concentrations of Zn and its deposition levels could be due to the factthat this metal is possibly present in different chemical forms depending on thesource of emission from which it is originated (for example ionic forms, metallicforms with an oxidation state of cero, organometallic compounds, etc.).


Figure 2. Lineal regression analysis between the total deposition of Cu and its content inT. 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 ratesin all the four species. Amongst them T. capillaris was the species with the highestvalues of a, indicating a greater efficiency of accumulation as also shown for Cuin Figure 2 and for Pb in Figure 3. As can be seen, differences in the efficiency ofaccumulation of most metals were found among the studied species, being highestin T. capillaris followed by T. permutata > T. tricholepis > T. retorta in decreasingorder. This efficiency could possibly be related to the anatomical features of eachspecies: for example, T. capillaris is the species with the greatest specific foliararea (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 atmosphericpollutants, the concentration of chemical physiological parameters was compared


Figure 3. Lineal regression analysis between the total deposition of Pb and its content inT. 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 thatthey were sensible to air pollution in other bioindicators such as lichens (Cunyet al., 2002) and plants (Wannaz et al., 2003).

For T. capillaris (Table IX), the samples that evidenced the greatest physiolog-ical 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 ofsulphur were also greatest in these areas. It is important to note that the associationof the accumulation of sulphur in a biomonitor with lipid peroxidation in mem-branes is a clear indication of damage due to air pollution, as previously describedfor lichens (Cuny et al., 2000). Furthermore, the content of sulphur in T. capillarisleaves significantly increased after 3 months compared to the basal values and waseven higher after 6 months exposure. It is interesting to state that the sulphur contentwas greater in the Southeast and Centre sites that are near emission sources of fossilderived fuels.


Table IX. Comparison of the mean values SD of the chemical parameters measured inT. capillaris. The values represent the result of a t-Test between the baseline sample andthe transplanted samples (3 and 6 months exposure) and the ANOVA between the differenttransplantation areas


Chl-aBaseline 0.340 0.020 0.340 0.020 0.340 0.020 0.340 0.0203 months 0.284 0.010 ab 0.187 0.027 b 0.222 0.018 ab 0.303 0.045 a 6 months 0.328 0.009 0.311 0.017 0.286 0.014 0.279 0.015 nst-Testa ns (0 = 3) (0 = 3) nsChl-a/Chl-bBaseline 0.416 0.009 0.416 0.009 0.416 0.009 0.416 0.0093 months 0.460 0.016 0.420 0.042 0.416 0.029 0.421 0.030 ns6 months 0.557 0.071 0.462 0.005 0.485 0.060 0.450 0.041 nst-Testa ns (0 = 6) ns nsMDABaseline 16.206 1.491 16.206 1.491 16.206 1.491 16.206 1.4913 months 33.829 4.131 30.861 0.619 33.122 1.867 32.786 2.834 ns6 months 30.880 8.472 ab 47.101 0.221 a 26.635 0.922 b 26.923 1.717 b t-Testa (0 = 3) (0 = 3; 0 = 6) (0 = 3; 0 = 6) (0 = 3; 0 = 6)HPCDBaseline 6.169 0.137 6.169 0.137 6.169 0.137 6.169 0.1373 months 10.644 2.710 10.578 1.693 8.649 1.009 8.939 1.791 ns6 months 8.995 0.717 9.247 2.054 7.888 1.195 7.261 2.621 nst-Testa (0 = 6) ns ns nsSulphurBaseline 0.155 0.137 0.155 0.137 0.155 0.137 0.155 0.1373 months 0.312 0.005 a 0.241 0.013 b 0.212 0.009 b 0.216 0.014 b 6 months 0.568 0.001 a 0.634 0.064 a 0.361 0.046 b 0.290 0.025 b t-Testa (0 = 3; 0 = 6) (0 = 3; 0 = 6) (0 = 3; 0 = 6) (0 = 3; 0 = 6)DW/FWBaseline 0.212 0.007 0.212 0.007 0.212 0.007 0.212 0.0073 months 0.243 0.017 0.235 0.010 0.234 0.007 0.236 0.020 ns6 months 0.541 0.092 0.510 0.111 0.486 0.049 0.423 0.088 nst-Testa (0 = 3; 0 = 6) (0 = 3; 0 = 6) (0 = 3; 0 = 6) (0 = 6)FDIBaseline 0.621 0.014 0.621 0.014 0.621 0.014 0.621 0.0143 months 2.293 0.432 1.596 0.242 1.481 0.048 1.437 0.048 ns6 months 7.680 1.057 a 10.255 1.739 a 2.788 0.664 b 4.005 0.850 b t-Testa (0 = 3) (0 = 3; 0 = 6) (0 = 3; 0 = 6) (0 = 3; 0 6)

ns, not significant; significant, P < 0.05; significant, P < 0.01; significant, P T. capillaris > T.retorta > T. permutata.

Finally, although not the object of the current investigation, it is important tonote the elevated levels of heavy metals (Cu, Ni, Fe, Co, Mn and Pb) found in thesamples of total atmospheric deposition in an industrialized area inside the city of


Cordoba. Furthermore, elevated levels of Zn were found in the centre of the cityand were even higher than those of the industrialized area. The presence of thismetal downtown is mainly associated with vehicular traffic and considering thehigh population density of the area these elevated levels of Zn are hazardous forphysiological response biomonitors the populations health.

Acknowledgements

This work was supported partially by the Agencia Nacional de Promocion Cientificay Tecnica (FONCyT), the Brazilian Synchrotron Light Laboratory (LNLS) andSecretaria de Ciencia y Tecnica de la Universidad Nacional de Cordoba (SECyT-UNC). We are especially grateful to Dr. Carlos A. Perez for his technical assistancein the measurement of metals with TXRF.

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Calibration of Four Species of Tillandsia as Air

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