Additional Information on Material and Methods

SUPPLEMENTARY MATERIAL

Additional information on Material and Methods

Environmental characteristics of the study area

The geological composition, the singular topographic and climatic conditions and ancestral human use have favored exceptional fauna and vegetal communities with a high diversity that represents one of the most remarkable natural areas in Catalonia. It is an area of climatic transition, with a confluence of typically Mediterranean elements or even some of African origin with other more septentrional. Altogether this favors the existence of high forest plant diversity in a relative small area. This natural area is now increasingly threatened by surrounding industries and cities.

Characteristics of the species studied

The species chosen to carry out this study were the moss Hypnum cupressiforme and the lichen Xanthoria parietina. These two species are widely distributed and abundant all across southern Europe (Nimis et al., 2001) and several studies have used them to determine trace element pollution (Scerbo, 1999; Brunialti and Frati, 2007). Some of such studies were carried out in Spain (Fernandez et al., 2000; Sardans and Peñuelas, 2005), which allows a comparison with previous studies in order to make a precise assessment of the pollution in the area.

Sampling process in each plot

The mosses were sampled from open areas and small forest clearings located as far as possible from the nearest tree (Rühling, 1994). The lichen samples were taken from trees at a height from 1.2 to 2.5 m and the maximum inclination for the thallus was 20%. Minimum size of the thallus was approximately 4 cm at the side. Only the thallus between the first apotecia and the outermost part was sampled (aprox. 5mm), given the difficulty of extracting exactly 3 mm in situ as described by Scerbo et al. (1999, 2002), and in order to collect Xanthoria parietina samples which had comparable timing to that of the moss samples, as that part of the lichen would represent the atmospheric deposition of the last 1-2 years. (Fisher and Proctor, 1978).

Analytical process

In the laboratory, the moss and lichen samples were cleaned to remove pieces of extraneous materials collected with the thalli during the field sampling. The upper green shoots (2 year-old parts) of the mosses were selected. The biomasses were cleaned because the aim of the study was to analyze the trace element concentrations in tissues in order to gain knowledge on the biological impact of pollution. Lichens and mosses cannot be washed with water since this can leach out significant amounts of different elements and alter the elemental composition of lichens (Bettinelli et al., 1996) and mosses (Bargagli, 1998). In order to clean the lichen and moss samples, we carefully brushed the samples and thereafter used a hair dryer to remove the fine particles (Sager and Mittendörfer, 1977). After all the samples had been cleaned, they were dried in an oven at 60 ºC to constant weight and then ground up using a ceramic mortar and pestle.

The soils were air dried for at least 72 hours. Thereafter, the biggest particles and aggregates were broken with a rolling pin, ground up with a ceramic mortar and pestle and sieved altogether in a 2 mm diameter sieve. We then analyzed the total soil trace element concentrations and also the soil concentrations of plant available trace elements by using two different extractants: H2O and 0.01M NaNO3. For soil extraction we took 2 g of soil and we added 12 mL of distilled water or 0.01M NaNO3 (Yin et al., 2002). The solutions were shaken for at least 12 hours in a shaking machine. After that, the sample was centrifuged for 10 minutes at 12000 rpm and 8 mL were taken to prepare the final sample.

An acid digestion of the samples was carried out by using the method EPA 3051a with HNO3 in a microwave oven (SAMSUNG TDS, Seoul, South Korea). Briefly, two mL of acid nitric (60%) were added to 100-125 mg of dry biomass for each sample. In every digestion set, a blank solution and a standard certified biomass (DC73351) were also digested in order to assess the accuracy of digestion and analytical procedures. To analyze biomass samples, dried and ground samples of lichen and moss were digested with concentrated HNO3 and H2O2 (30%. p/v) (MERCK, Darmstadt, Germany) in a microwave oven. Blank solutions were regularly analyzed. A standard certified biomass (NIST 1573a leaf tomato, NIST, Gaitherburg, MD) was used to assess the accuracy of digestion and the analytical biomass procedures.

The concentrations of Al, Ti, Pb, Cr, Cd, Ni, As, Co, Sb, Zn, Cu and V were determined using ICP-MS (Mass Spectrometry with Inductively Coupled Plasma) in the acid digested solutions and soil extracts.

Table S1. Soil concentrations (mg g-1, except for Al: mg g-1). The bold type indicates the maximum values and the italic bold type stands for the minimum values.

Sample / Al* / V / Cr / Co / Ni / Cu / Zn / As / Cd / Pb
Alforja / 21.7 / 60.1 / 9.9 / 6.6 / 3.4 / 7.3 / 52.4 / <2.5 / 0.06 / 11.6
Febro / 25.4 / 22.6 / 26.0 / 6.1 / 18.9 / 11.0 / 25.2 / 5.3 / 0.12 / 12.1
Siurana / 12.7 / 29.6 / 13.8 / 4.7 / 12.0 / 7.9 / 30.0 / 6.1 / 0.10 / 16.8
Perrot / 14.5 / 45.2 / 37.2 / 12.7 / 23.7 / 21.2 / 31.3 / 6.6 / 0.09 / 21.9
Prades / 9.9 / 16.4 / 13.9 / 4.8 / 10.4 / 13.2 / 25.2 / 5.7 / 0.06 / 8.4
Vilanova / 12.4 / 23.6 / 15.9 / 4.2 / 10.0 / 8.5 / 31.3 / 8.6 / 0.22 / 17.4
Montblanc / 17.8 / 34.4 / 19.8 / 6.3 / 14.7 / 15.3 / 41.6 / 14.6 / 0.14 / 34.0
Pena / 33.1 / 51.3 / 12.8 / 9.0 / 7.3 / 24.2 / 342.3 / 11.9 / 1.10 / 87.3
Poblet / 19.3 / 53.5 / 22.1 / 9.0 / 26.6 / 52.8 / 176.1 / 23.4 / 0.63 / 56.8
Rojalons / 14.7 / 25.7 / 16.7 / 7.3 / 16.6 / 15.9 / 43.3 / 13.2 / 0.18 / 27.5
Rojals / 16.5 / 26.7 / 15.3 / 5.2 / 12.9 / 11.7 / 36.6 / 7.6 / 0.19 / 18.0
Alcover / 17.1 / 23.9 / 18.3 / 7.0 / 16.7 / 25.3 / 39.7 / 10.0 / 0.16 / 15.5
Gomis / 17.1 / 25.3 / 17.0 / 6.3 / 16.0 / 47.8 / 36.9 / 10.1 / 0.22 / 16.9
Montral / 18.8 / 27.0 / 20.7 / 6.6 / 16.3 / 14.1 / 32.9 / 8.5 / 0.12 / 22.6
Pinetell / 28.6 / 45.9 / 28.4 / 9.0 / 24.0 / 19.7 / 40.0 / 26.7 / 0.25 / 47.6
Riba / 17.7 / 21.7 / 17.0 / 6.4 / 16.3 / 24.9 / 52.5 / 7.8 / 0.16 / 15.0
MIN / 9.9 / 16.4 / 9.9 / 4.2 / 3.4 / 7.3 / 25.2 / 5.3 / 0.06 / 8.4
MAX / 33.1 / 60.1 / 37.2 / 12.7 / 26.6 / 52.8 / 342.3 / 26.7 / 1.10 / 87.3
MEAN / 18.6 / 33.3 / 19.0 / 7.0 / 15.4 / 20.0 / 64.8 / 11.1 / 0.24 / 26.8
MEDIAN / 17.4 / 26.8 / 17.0 / 6.5 / 16.1 / 15.6 / 38.3 / 8.6 / 0.16 / 17.7

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Table S2. Correlation coefficients between the distance from the local pollution sources to the different sampling locations and trace element concentrations (mg g-1). The bold type is for significant correlations (p<0.05) and the italics are for marginally significant correlations.

Biomonitor / Element / Riba / Montblanc / Picamoixons / Pobla / Tarragona / Alcover
Hypnum
cupressiforme / Cu / R=-0.45 / R=-0.25 / R=-0.47 / R=-0.38 / R=-0.25 / R=-0.35
p=0.073 / p=0.353 / p=0.067 / p=0.146 / p=0.368 / p=0.185
Zn / R=-0.49 / R=-0.22 / R=-0.52 / R=-0.51 / R=-0.40 / R=-0.52
p=0.057 / p=0.402 / p=0.045 / p=0.043 / p=0.135 / p=0.042
Sb / R=-0.48 / R=-0.13 / R=-0.51 / R=-0.52 / R=-0.41 / R=-0.54
p=0.060 / p=0.634 / p=0.044 / p=0.047 / p=0.124 / p=0.033
Xanthoria parietina / Cr / R=-0.30 / R=0.20 / R=-0.42 / R=-0.65 / R=-0.61 / R=-0.65
p=0.352 / p=0.547 / p=0.173 / p=0.021 / p=0.035 / p=0.024
Ni / R=-0.33 / R=0.18 / R=-0.45 / R=-0.67 / R=-0.62 / R=-0.66
p=0.303 / p=0.575 / p=0.142 / p=0.017 / p=0.032 / p=0.020
Sb / R=-0.19 / R=0.20 / R=-0.29 / R=-0.52 / R=-0.52 / R=-0.42
p=0.554 / p=0.534 / p=0.357 / p=0.082 / p=0.083 / p=0.136

Table S3. Correlation coefficients between the distances from the local pollution sources to the different sampling locations and Factor analysis (FA) scores of the different plots of the FA conducted with trace elements concentrations as variables and sampling locations as cases. X is for Xanthoria parietina. H is for Hypnum cupressiforme. The bold type is for significant correlations (p<0.05) and the italics are for marginally significant correlations.

Riba / Montblanc / Picamoixons / Pobla / Tarragona / Alcover / Metro. Area / Camp / Summatory
X/F1 / R=-0.07 / R=-0.30 / R=0.01 / R=0.26 / R=0.34 / R=0.25 / R=0.30 / R=0.06 / R=0.07
p=0.613 / p=0.282 / p=0.987 / p=0.355 / p=0.224 / p=0.376 / p=0.275 / p=0.846 / p=0.821
X/F2 / R=-0.69 / R=-0.26 / R=-0.73 / R=-0.76 / R=-0.62 / R=-0.78 / R=-0.71 / R=-0.77 / R=-0.78
p=0.005 / p=0.35 / p=0.002 / p=0.001 / p=0.014 / p>0.001 / p=0.003 / p=0.001 / p<0.001
H/F1 / R=0.06 / R=0.10 / R=-0.05 / R=-0.05 / R=-0.08 / R<-0.01 / R=-0.07 / R=0.04 / R=-0.03
p=0.824 / p=0.732 / p=0.851 / p=0.866 / p=0.778 / p=0.991 / p=0.814 / p=0.885 / p=0.923
H/F2 / R=-0.17 / R=0.04 / R=-0.25 / R=-0.38 / R=-0.35 / R=-0.40 / R=-0.37 / R=-0.27 / R=-0.29
p=0.552 / p=0.887 / p=0.377 / p=0.169 / p=0.217 / p=0.143 / p=0.175 / p=0.322 / p=0.301

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Table S4. Correlation coefficients between the distances from sampling locations to the local pollution sources and EF values. The bold numbers indicate significant correlations (p<0.05) and the italic numbers marginally significant correlations (0.05 < p < 0.1).

Biomonitor / Trace element / Riba / Montblanc / Picamoixons / Pobla / Tarragona / Alcover
Hypnum cupressiforme / V / R=-0.58 / R<0.01 / R=-0.67 / R=-0.81 / R=-0.76 / R=-0.75
p=0.023 / p=0.992 / p=0.006 / p<0.001 / p=0.001 / p=0.001
Zn / R=-0.56 / R=-0.02 / R=-0.64 / R=-0.76 / R=-0.75 / R=-0.74
p=0.029 / p=0.958 / p=0.010 / p=0.001 / p<0.001 / p=0.002
Cd / R=-0.33 / R=0.01 / R=-0.38 / R=-0.45 / R=-0.43 / R=-0.36
p=0.239 / p=0.964 / p=0.162 / p=0.092 / p=0.112 / p=0.195
Pb / R=-0.21 / R=0.28 / R=-0.29 / R=-0.49 / R=-0.52 / R=-0.38
p=0.457 / p=0.323 / p=0.302 / p=0.062 / p=0.048 / p=0.164
Xanthoria parietina / V / R=-0.45 / R=0.06 / R=-0.51 / R=-0.67 / R=-0.66 / R=-0.59
p=0.117 / p=0.844 / p=0.065 / p=0.010 / p=0.010 / p=0.028
Cr / R=-0.43 / R=-0.09 / R=-0.48 / R=-0.56 / R=-0.54 / R=-0.59
p=0.123 / p=0.755 / p=0.082 / p=0.036 / p=0.046 / p=0.026
Co / R=-0.34 / R=0.08 / R=-0.42 / R=-0.63 / R=-0.66 / R=-0.64
p=0.234 / p=0.787 / p=0.14 / p=0.016 / p=0.011 / p=0.014
Ni / R=-0.45 / R=-0.16 / R=-0.49 / R=-0.52 / R=-0.48 / R=-0.56
p=0.103 / p=0.591 / p=0.078 / p=0.058 / p=0.085 / p=0.037

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Table S5. Correlation coefficients between the distances from the local pollution sources to the different sampling locations and PC scores of the two first axes of the FA conducted with trace element enrichment factors as variables and sampling locations as cases. The lower case “e” indicates that only the 5 most enriched trace element concentrations (mg g-1) were used. The bold type is for significant correlations (p<0.05) and the italics are for marginally significant correlations.

Riba / Montblanc / Picamoixons / Pobla / Tarragona / Alcover / Metro. Area / Camp / Summatory
XEF/F1 / R=0.21 / R=0.44 / R=0.17 / R=-0.13 / R=-0.28 / R=0.03 / R=-0.21 / R=0.14 / R=0.13
p=0.464 / p=0.102 / p=0.543 / p=0.620 / p=0.325 / p=0.907 / p=0.459 / p=0.606 / p=0.655
XEF/F2 / R=-0.30 / R=-0.03 / R=-0.36 / R=-0.53 / R=-0.51 / R=-0.54 / R=-0.53 / R=-0.41 / R=-0.44
p=0.275 / p=0.921 / p=0.182 / p=0.043 / p=0.048 / p=0.045 / p=0.043 / p=0.132 / p=0.100
HEF/F1 / R=-0.21 / R=-0.12 / R=-0.23 / R=-0.12 / R=-0.01 / R=-0.19 / R=-0.07 / R=-0.22 / R=-0.19
p=0.461 / p=0.673 / p=0.402 / p=0.660 / p=0.979 / p=0.485 / p=0.803 / p=0.423 / p=0.495
HEF/F2 / R=-0.17 / R=0.26 / R=-0.26 / R=-0.56 / R=-0.60 / R=-0.39 / R=-0.59 / R=-0.28 / R=-0.32
p=0.545 / p=0.367 / p=0.355 / p=0.032 / p=0.027 / p=0.145 / p=0.026 / p=0.310 / p=0.254
XEF/F1e / R=0.28 / R=0.39 / R=0.24 / R=-0.03 / R=-0.17 / R=0.04 / R=-0.10 / R=0.20 / R=0.19
p=0.392 / p=0.395 / p=0.394 / p=0.396 / p=0.391 / p=0.396 / p=0.395 / p=0.476 / p = 0.503
XEF/F2e / R=-0.36 / R=-0.05 / R=-0.42 / R=-0.57 / R=-0.54 / R=-0.55 / R=-0.56 / R=-0.46 / R =-0.49
p=0.186 / p=0.857 / p=0.120 / p=0.033 / p=0.042 / p=0.036 / p=0.031 / p=0.086 / p=0.060
HEF/F1e / R=0.22 / R=-0.08 / R=0.29 / R=0.39 / R=0.33 / R=0.31 / R=0.37 / R=0.28 / R=0.28
p=0.439 / p=0.767 / p=0.294 / p=0.154 / p=0.222 / p=0.261 / p=0.183 / p=0.312 / p=0.310
HEF/F2e / R=0.46 / R=0.32 / R=0.40 / R=0.15 / R=-0.02 / R=0.16 / R=0.070 / R=0.36 / R=0.33
p=0.088 / p=0.256 / p=0.147 / p=0.595 / p=0.932 / p=0.573 / p=0.811 / p=0.189 / p=0.230

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Table S6. Correlation coefficients between the distances from sampling locations to local pollution sources and lichen richness values. The bold numbers are for significant correlations (p<0.05) and the italic numbers are for marginally significant correlations.

Variable / Riba / Montblanc / Picamoixons / Pobla / Tarragona / Alcover / Metro. Area / Camp / Summatory
Lichens/tree / R=0.46 / R=0.08 / R=0.52 / R=0.63 / R=0.57 / R=0.53 / R=0.61 / R=0.52 / R=0.55
p=0.085 / p=0.784 / p=0.050 / p=0.011 / p=0.027 / p=0.045 / p=0.015 / p=0.048 / p=0.033
Lichen richness / R=0.55 / R=0.19 / R=0.59 / R=0.66 / R=0.56 / R=0.59 / R=0.62 / R=0.60 / R=0.63
p=0.034 / p=0.501 / p=0.020 / p=0.008 / p=0.028 / p=0.020 / p=0.013 / p=0.018 / p=0.011

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Table S7. Comparison of means and medians of trace element concentrations (mg g-1) of this study with those of various studies carried out using Hypnum cupressiforme and Xanthoria parietina.

Study / Value / Al / Ti / V / Cr / Co / Ni / Cu / Zn / As / Cd / Sb / Pb
Hypnum cupressiforme / This styudy / Mean / 3408 / 68.7 / 3.07 / 15.06 / 0.52 / 8.42 / 6.66 / 28.81 / 0.41 / 0.13 / 0.43 / 2.73
This study / Median / 2585 / 38.3 / 2.67 / 12.14 / 0.46 / 7.53 / 6.22 / 28.65 / 0.38 / 0.11 / 0.4 / 2.38