Atmospheric Environment 43 (2009) 5501–5509

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Atmospheric Environment
journal homepage: www.elsevier.com/locate/atmosenv

Use of the terrestrial moss Pseudoscleropodium purum to detect sources

of small scale contamination by PAHs
A. Ares a, *, J.R. Aboal a, J.A. Fernández a, C. Real b, A. Carballeira a
a
Ecologı́a, Facultad de Biologı́a, Universidad de Santiago de Compostela, 15782 Santiago de Compostela, Spain
b
Ecologı́a, Escuela Politécnica Superior, Universidad de Santiago de Compostela, 27002 Lugo, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Here we present a simple, economic method of identifying sources of small scale contamination by
Received 12 March 2009 polycyclic aromatic hydrocarbons (PAHs). The method involves determining the concentrations of the
Received in revised form contaminants in the terrestrial moss Pseudoscleropodium purum and consists of the following steps: i)
2 June 2009
testing for the existence of gradients of decreasing concentrations of PAHs in the moss in relation to
Accepted 6 July 2009
distance from different emission sources; ii) measurement of the concentration of PAHs at 35 pairs of
sampling sites, each separated by a distance of 1 km; iii) study of the distribution of the differences in
Keywords:
concentration between these pairs of sampling sites and elimination of extreme values (affected by small
Polycyclic aromatic hydrocarbons
Pseudoscleropodium purum scale sources of contamination); iv) characterization of normal distributions to determine the probability
Air quality of the data being thus distributed; and v) testing the method in the surroundings of possible sources of
Contamination small scale contamination by PAHs. The decrease in concentration of all of the compounds followed
a steep gradient with increasing distance from the emission source; after elimination of the outliers, the
distribution of the differences in concentration between the 35 pairs of sampling sites was normal for all
compounds, except benzo(a)pyrene. Application of the method to 15 different types of industries
provided satisfactory results and the method proved to be a very useful tool for monitoring and eval-
uating air quality.
Ó 2009 Elsevier Ltd. All rights reserved.

1. Introduction stations, domestic heating, burning rubbish dumps, tobacco smoke,

Terrestrial mosses have been used for monitoring air quality
ever since the second half of the 20th century, and the first studies
were carried out with heavy metals (Rühling and Tyler, 1968;
Goodman and Roberts, 1971). However, mosses were not used to
biomonitor air quality as regards organic microcontaminants until
the 1980s (Thomas, 1979; Wegener et al., 1991), even though such
compounds are highly toxic. Organic contaminants include poly-
cyclic aromatic hydrocarbons (PAHs), seven of which have been
classified as possible human carcinogens by the US Environment
Protection Agency (EPA, 1993) (i.e. benz(a)anthracene, benzo(a)-
pyrene, benzo(b)fluoranthene, benzo(k)fluoranthene, dibenzo(a,-
h)anthracene, crysene and indeno(1,2,3-c,d)pyrene). Furthermore,
these contaminants are widely distributed in the atmosphere and
have diverse origins. They can be emitted by natural processes (e.g.
volcanic eruptions, wild fires, etc.), and also by anthropogenic
processes, principally those derived from the incomplete combus-
tion of fossil fuels (Currie, 2000) (e.g. in vehicles, power generating
* Corresponding author.
E-mail address: aa.pita@gmail.com (A. Ares).
etc.). Two approaches have been applied in using terrestrial mosses
to study air quality as regards PAHs: intensive and extensive
studies. In intensive studies, the contamination is examined in the
surroundings of small scale contamination foci in industrialized
areas, by the use of dense sampling networks. This enables evalu-
ation of the magnitude and extent of the contamination in the
surroundings of industrial complexes, factories, areas of heavy
traffic, etc. (see for example: Wegener et al., 1991; Viskari et al.,
1997; Orliński, 2002; Bignal et al., 2007). On the other hand,
extensive studies are carried out in large areas, by the use of
sampling networks, with the aim of evaluating patterns of large
scale contamination or of evaluating the air quality in a particular
region (see for example: Carlberg et al., 1983; Thomas and Schunke,
1984; Galuszka, 2007; Holoubek et al., 2007).
However, until now no specific method has been recommended
for the detection and identification of the PAHs emitted by each
different source of small scale contamination that may be located
over wide areas. The intensive study of each source, as described
above, would be extremely costly and impractical. An extensive
study would not be appropriate either, as many of the sources
would be overlooked because of the density of sampling sites (SS)
used in this type of network. Although no specific method has been

1352-2310/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.atmosenv.2009.07.005
5502 A. Ares et al. / Atmospheric Environment 43 (2009) 5501–5509

described for the detection of sources of small scale contamination

by PAHs, a method has been proposed with the same objectives but
B 1.V
A
for the detection of small scale contamination by heavy metals and B
metalloids (Fernández et al., 2007). The aim of the present study Spain

was to develop a similar method to previously proposed method for

the rapid and economic detection of small scale sources of
1.IV
contamination by PAHs, because of the high potential toxicity and
great environmental persistence of these compounds (Yu, 2005). 1.III
0 350 700 1.400 2.100 2.800
The method is based on how the contaminants are bio- 1.II
1.I b Meters
accumulated in the surroundings of the source of contamination. c
a 2.I
Previous studies with other types of contaminants have shown that 2.II 2.III
2.IV
2.V
the decrease in the concentration of some contaminants in moss
with increasing distance from the source of emission follows an
exponential distribution. The concentrations thus decrease rapidly
A c d
b a e
0 350 700 1.400 2.100 2.800
in the vicinity of the source, as shown in numerous studies of heavy Meters 1II 1.I
2.I
f
2.II
metals (see for example: Türkan et al., 1995; Fernández et al., 2000; 1.III 2.III
1.IV
Pöykiö et al., 2001; Real et al., 2003). Thus the differences in
concentrations of the contaminant in a sample of moss collected in 2.IV

the immediate surroundings of the source and another collected at

a point further from the source will generally be large, indicating
that the source contaminates the immediate surroundings. 1.V

However, very few studies of contamination by PAHs have shown

this type of distribution (Herrmann and Hübner, 1984; Viskari et al.,
1997).
In order for the proposed method to function, we first had to
verify that the differences in concentration of elements between
a moss sample collected in the immediate vicinity of the source and
another collected at a distance from the source were due to the
presence of contamination and not simply to variability in the data.
To test this, we established the distribution of the differences in
concentration between pairs of samples separated by a short
distance in zones that are assumed to be free from contamination
by PAHs. We then determined whether or not values corresponding
to areas close to the sources belong to this distribution; otherwise,
we suspected that the source contaminated the surrounding area.
One important point to consider is the distance between each
SS, as this must be sufficiently large to include the contamination
gradient, but not too large so that the gradient is not affected by
large scale contamination foci. Taking into account the area affected
by sources of small scale contamination by heavy metals (i.e.
a radius of between 4 and 8 km according to Fernández et al., 2007),
a distance of 1 km was proposed as a compromise between the two
requirements.
The objectives of the present study were therefore: i) to test for
the existence of an exponential decrease in the concentration of the
different PAHs in moss with increasing distance from an emission
source; ii) to establish the distribution of differences in concen-
tration of each PAH in pairs of SS sampled in areas not affected by
sources of small scale contamination, and iii) to apply the method
to potential sources of small scale emission of PAHs.
2. Material and methods
2.1. Sampling
The study was carried out in Galicia (NW Spain) (Fig. 1); the
species selected for study was the bryophyte Pseudoscleropodium
purum (Hedw.) M. Fleisch. All of the samples were collected, when
possible, at distances of more than 300 m from main roads and
populated areas, and at least 100 m from other types of roads and
isolated houses. Whenever possible the samples were collected in
open areas, and when this was not possible, in forest clearings. The
areas where the moss samples were collected at each SS were no
larger than 50  50 m and no smaller than 20  20 m. As recom-
mended by Fernández et al. (2002) and Aboal et al. (2006),
N 2.V
Fig. 1. Maps showing the geographical location of the sampling area (inset) (Galicia,
NW Spain), as well as the factories under study (A and B) and the two transects (1 and
2) at each factory. Circles represent possible contamination foci, triangles (factory A)
and squares (factory B) represent the points where moss samples were collected.
subsamples were collected (in this case at least 30 subsamples) and
were grouped into composite samples.
2.1.1. Characterization of PAH gradients in industrial areas
Firstly, in order to determine the pattern of decrease in the
concentration of PAHs in the moss with increasing distance from
the emission source, two factories assumed to generate small scale
contamination with these compounds were selected for study.
Factory A is an aluminium smelter and factory B is an Fe–Mn
smelter; both use coke and fuel oil and presumably emit PAHs into
the atmosphere. Taking into account the situation of the principal
emission foci in each factory, two transects were designed (Fig. 1),
each comprising 5 SS, positioned at increasing distances from each
factory. In factory A, transect 1 ran from the presumed principal
source of emission ‘‘a’’ and transect 2 from another source ‘‘e’’,
whereas in factory B, the transects were positioned almost
perpendicular to each other, from the position of all of the emission
sources (‘‘a, b and c’’). The samples were collected in March 2007.
2.1.2. Estimation of the distributions of the differences in
concentration between pairs of SS
A total of 35 SS were selected, 27 of which corresponded to 2
samplings carried out in March and September 2004 for the Gali-
cian Air Quality Biomonitoring Network. These samples were
previously used by Fernández et al. (2007) to monitor heavy metals
and metalloids. In the present study, frozen samples (30  C) were
used wherever possible (11 pairs), but when more material was
required, dried samples stored at room temperature (20  C) were
used (16 pairs). Both samples in each pair were always preserved in
the same way. The remaining 8 pairs were obtained in the sampling
described in the previous section and when the pairs of SS were
separated by the distance required. These samples were stored at
room temperature until analysis. In all of the previous cases, the
mean distance between the SS in each pair was 1114  143 m
(mean  95% confidence interval).
 A. Ares et al. / Atmospheric Environment 43 (2009) 5501–5509
The pairs of sampling sites corresponding to the sampling
carried out in 2004 in the Galician Air Quality Biomonitoring
Network were identified with the corresponding code and indi-
cating the month in which the sampling took place (m: March, s:
September). In the case of factories A and B, the following pairs of
SS were selected (Fig. 1): A1 (comprising SS A1I þ A1IV), A2
(A1II þ A2II), A3 (A2I þ A2IV), A4 (A1III þ A2III), B1 (B1I þ B1IV), B2
(B1II þ B2II), B3(B2I þ B1III) and B4 (B2II þ B2IV).
2.1.3. Application of the method in the vicinity of sources of
contamination
In order to develop the proposed method, 15 factories that
potentially generate small scale contamination by PAHs were
selected within the study region. Samples were collected from
along two radial transects from the presumed source of emission,
each comprising two SS separated by a distance of approximately
1 km.
The factories sampled – in addition to those described in Section
2.1.1 – were: a cement works, three ceramic factories, a glass
factory, three fibreboard factories, two iron smelters (one of these
was sampled on two occasions, once before and once after tech-
nological alterations aimed at decreasing the emission of contam-
inating compounds), a biodiesel plant, a waste oil treatment plant
and a cogeneration plant.
Sampling was carried out in 2004 and in 2008, except at the iron
smelter analysed before the above-mentioned technological alter-
ations, which was sampled in 2002.
2.2. Sample processing
In the laboratory, the apical segments (3–4 cm) of the moss
samples were separated from the shoots. Adhered material (plant
remains, soil particles, etc.) was removed manually. To ensure the
integrity of the membrane, the moss was acclimatised (exposed to
a moisture-saturated atmosphere at 10  C) for 7 days, then washed
for 30 s with bidistilled water – with shaking – dried and finally
ground in an ultracentrifuge mill (Retsch ZM100).
As the samples used to estimate the differences in concentration
between the pairs of SS were stored in two different ways (at room
temperature after drying, or frozen at 30  C), some samples were
analysed to establish if the PAHs were conserved equally well with
both methods. For this purpose, 5 pairs of samples of which there
was sufficient material stored in both ways were analysed. The
frozen samples were dried, ground and processed in June 2007, in
the same way as the dried samples.
2.3. Chemical analysis
Determination of the concentration of the PAHs was carried out
at the Instituto de Medio Ambiente (University of La Coruña). For
extraction of PAHs, between 0.6 and 0.8 g of moss were weighed
and added to a solution of deuterized PAH standards dissolved in
acetone (naphthalene d-8, acenaphthene d-10, phenanthrene d-10
and crysene d-12 (Supelco)). Thirty milliliter of a mixture of
hexane/acetone (1:1) were then added (n-hexane for trace organic
analysis (Merck) and acetone, super purity solvent (Romil)).
For extraction, the samples were placed in a microwave oven
(ETHOS SEL, Milestone) for 30 min at 130  C. The extracts were
filtered with glass fibre filters (MN GF 6: Macherey-Nagel, diameter
5.5 cm and thickness 0.35 mm) along with 9 mL of the hexane/
acetone mixture (1:1).
To purify the samples, glass columns were filled with silanized
glass wool and 12 g of florisilÒ (60–100 mesh, Aldrich) and 4 g of
silica gel (70–230 mesh, Sigma) were added (after prior inactivation
5503
by addition of water (5%)). Finally, 0.5 g of granulated anhydrous
sodium sulphate for organic trace analysis (Merck) was added.
The adsorbents used were previously washed in the Soxhlet
extractor with a mixture of dichloromethane/methanol (2:1)
(dichloromethane, super purity solvent Romil); methanol, super
purity solvent (Romil), for 12 h and then for another 12 h with
a mixture of dichloromethane/hexane (30:70).
Once dried, the silica gel was activated at 130  C, the florisil at
350  C and the anhydrous sodium sulphate at 100  C, for 12 h. After
being washed and activated, the adsorbents were stored in
a desiccator until further use.
In order to remove the dissolvent, the extracts were concen-
trated in a rotary evaporator at 32  C to the volume of a drop. The
drop was then loaded on the column and eluted with 45 mL of
a mixture of dichloromethane/hexane (20:80). The eluate was then
concentrated in the rotary evaporator to a drop, which was dried in
a stream of N2 at a flow rate of 60 mL min1.
The PAHs were analysed by gas chromatography-mass spec-
trometry in MS/MS mode (Column: J&W, DB-XLB 60 m  0.25
mm  0.25 mm. Injection was in PTV mode, with a temperature
programme. Initial temperature was 50  C, speed of transfer,
3.3  C s1, final temperature 300  C [20 min]. Injection volume was
7 mL, temperature, 50  C (4 min), 6  C min1 to 200  C, 4  C min1 to
325  C [10 min]).
Deuterized PAH standards were used as reference material as
there is no material similar to moss available for determination of
these compounds.
The PAHs determined were: naphthalene, acenaphthene, fluo-
rene, phenanthrene, anthracene, fluoranthene, pyrene, benz(a)an-
thracene, crysene, benzo(b)fluoranthene þ benzo(j)fluoranthene,
benzo(k)fluoranthene, benzo(e)pyrene, benzo(a)pyrene, benzo(g-
hi)perylene, dibenz(ah)anthracene, and indenopyrene. The limits of
quantification varied between 9.1E05 for benz(a)anthracene,
crysene, benzo(b)fluoranthene þ benzo(j)fluoranthene, benzo(k)-
fluoranthene, benzo(e)pyrene, benzo(a)pyrene, benzo(ghi)per-
ylene, dibenz(ah)anthracene and indenopyrene, and 0.014 ng g1
for naphthalene. Some of the concentrations of benzo(a)pyrene
determined in estimating the distribution of the differences in
concentration between the pairs of SS (Section 2.1.2) were below
the limit of quantification, and therefore to be able to carry out the
statistical analysis of the data, the values were assumed to be half of
this value. The mean percentage recovery was higher than 90% for
all of the PAHs except naphthalene, for which it was 60%.
2.4. Statistical treatment
In order to test for any significant differences between the
methods of storing the samples (at room temperature after drying,
and freezing at 30  C), a Wilcoxon’s test for related samples was
applied (p < 0.01). A Mann–Whitney U test was used to compare
the median concentrations of the pollutants in the surveys carried
out in March and September 2004. A Spearman’s correlation test
was used to establish whether there was any relationship between
the mean concentration corresponding to each pair of S.S. and the
magnitude of the differences between both. The normality of the
distribution of the differences in concentration between the pairs
of samples was analysed by a Shapiro–Wilks test (n ¼ 35). The tests
were carried out with SPSS version 15.0.
Kernel smoothing was used to estimate the density distribution
of the data for each distribution, (Wand and Jones, 1995). A
Gaussian kernel was selected and the width was optimized for each
set of data using the direct introduction technique with two levels
of functional estimation. The calculations were made with the
KernSmooth package (Ripley, 2002) in R (R Development Core
Team, 2004).
5504 A. Ares et al. / Atmospheric Environment 43 (2009) 5501–5509

600 40 40 600
Naphthalene Acenaphthene Fluorene Phenanthrene

450 30 30 450

300 20 20 300

150 10 10 150

0 0 0 0

200 2000 1200 100

Anthracene Fluoranthene Pyrene Benz(a)anthracene

150 1500 900 75

100 1000 600 50

50 500 300 25
Concentration (ng g-1)

0 0 0 0
200 120 40 60
Crysene Benzo(b)fluoranthene+ Benzo(k)fluoranthene Benzo(e)pyrene
Benzo(j)fluoranthene

150 90 30 45

100 60 20 30

50 30 10 15

0 0 0 0
36 12 80 80
Benzo(a)pyrene Benzo(ghi)perylene Dibenz(ah)anthracene Indenopyrene

27 9 60 60

18 6 40 40

9 3 20 20

0 0 0 0
0 1000 2000 3000 4000 0 1000 2000 3000 4000 0 1000 2000 3000 4000 0 1000 2000 3000 4000
Distance from source (m)

Fig. 2. Relation between the concentration of the contaminant in the moss Pseudoscleropodium purum (ng g1) and the distance from the focus of contamination (m) at the two
factories (A and B) and in both transects, for each of the compound analysed. Triangles represent the sampling points at factory A (white triangles: transect 1; black triangles:
transect 2). Squares represent the sampling points at factory B (white squares: transect 1; black squares: transect 2).
 A. Ares et al. / Atmospheric Environment 43 (2009) 5501–5509 5505

25
4800
Total PAHs Total PAHs

Probability (X10-2)
Concentration (ng g-1)

20
3600

15

p<0.001
p<0.01
p<0.05
2400

10
1200

5
A2 B1 A1
B3

0
0
0 1000 2000 3000 4000 0 1000 2000 3000
Distance from source (m) Differences in concentration

Fig. 3. a) Relation between the total concentration of all of the PAHs in the moss Pseudoscleropodium purum (ng g1) and distance from the focus of contamination (m) for both
factories (A and B) and both transects. b) Kernel-smoothing estimates of the distributions of the differences in the concentrations in the moss Pseudoscleropodium purum between
pairs of sampling sites (SS) separated by a distance of 1 km, for the sum of all of the PAHs. The values of the distribution are shown on the secondary x axis. In this case the
differences that must exist between a pair of SS to consider them as being outside of the distribution, at different levels of significance, are also shown.

3. Results No prior information was available as regards the levels of

3.1. Characterization of PAH gradients in industrial areas
The variation in the concentration of PAHs in samples of P. purum
with increasing distance from the emission source, for transects 1
and 2 and factories A and B (1A, 2A; 1B, 2B) is shown in Fig. 2.
At both factories and in both transects, the concentration of PAHs
decreased with increasing distance from the emission source, although
the decrease in concentration was more marked in transect 1 than in
transect 2 at both factories (Fig. 2). In 1A there were clear exponential-
type gradients of decreasing contamination for some compounds (e.g.
phenanthrene, anthracene, fluoranthene, etc.), whereas in 1B the same
pattern of decrease was observed for other compounds (e.g. ace-
naphthene, fluorene, benzo(b)fluoranthene þ benzo(j)fluoranthene,
etc). However, an anomalous increase in concentrations of some of the
compounds (e.g. benzo(a)pyrene and indenopyrene) was found in 1A
and at point 4. The pattern of decreasing concentration was not as clear
in 2A and 2B as in 1A and 1B.
The concentration of all 16 PAHs decreased sharply in transect 1
at both factories, especially factory A (Fig. 3). The greatest differ-
ences in concentration occurred between points 1 and 4 in both
transects at both factories (especially in 1A and 1B), which confirms
that a distance of 1 km between the SS was sufficient for sample
collection.
3.2. Differences between frozen samples and dried samples stored
at room temperature
contamination of PAHs in the zones that may be affected by
contamination foci. To resolve this problem, a series of steps was
followed in order to identify and disregard those pairs assumed to
be affected by small scale contamination processes: i) use of kernel
smoothing to construct the distributions; ii) identification of the
extreme values of the distributions; iii) search for possible foci that
affect the extreme values; iv) elimination of extreme values and v)
validation of the normality of the resulting distributions.
The graphs of the distribution of the differences in concentra-
tion, as well as the extreme values eliminated for each of the PAHs
studied are shown in Fig. 4.
After eliminating the extreme values, all of the compounds were
found to be normally distributed, except for benzo(a)pyrene. For all of
the distributions, most of the extreme values corresponded to pairs of
SS in the industrial areas A and B, i.e. for: naphthalene, acenaphthene,
phenanthrene, pyrene, benzo(b)fluoranthene þ benzo(j)fluoranthene,
benzo(k)fluoranthene, benzo(e)pyrene, benzo(ghi)perylene, diben-
z(ah)anthracene, indenopyrene and the sum of PAHs. It was assumed
that none of the pairs of SS from the samplings carried out in 2004
within the Galician Air Quality Biomonitoring Network were within
the area of influence of a source of contamination by these
Table 1
Median concentrations of PAHs (ng g1) obtained for the same samples (n ¼ 5) of
Pseudoscleropodium purum stored at 30  C or at room temperature (20  C) for
a period of 4 years, and the result of the application of Wilcoxon’s test (Z value).
PAH Median Z value Significance

There were no significant differences (p < 0.01) between the 30  C Room

two types of storage treatments for any of the compounds deter- temperature
mined, nor for the total PAHs (Table 1). For those compounds for Naphthalene 222 300 1.214 0.225
which significant differences were observed at p < 0.05, the Acenaphthene 3.15 3.09 0.944 0.345
Fluorene 4.46 3.98 0.405 0.686
concentrations in the samples stored at room temperature were Phenanthrene 14.1 19.8 1.753 0.080
higher than those in the frozen samples. Anthracene 1.25 2.99 0.944 0.345
Fluoranthene 5.96 9.20 2.023 0.043
Pyrene 4.08 6.80 2.023 0.043
3.3. Distributions of the differences in concentration of the pairs of Benz(a)anthracene 2.08 1.18 0.365 0.715
sampling sites Crysene 3.06 4.65 1.753 0.080
Benzo(b)fluoranthene þ 2.16 3.16 2.023 0.043
Benzo(j)fluoranthene
There were no significant differences (p < 0.05) between the
Benzo(k)fluoranthene 0.563 0.910 1.483 0.138
median values for the two surveys carried out (March and Benzo(e)pyrene 0.929 1.96 2.023 0.043
September), and therefore the data were pooled to form a single Benzo(a)pyrene 9.09E05 9.09E05 1.000 0.317
data set. The magnitude of the difference in concentrations for each Benzo(ghi)perylene 0.364 9.09E05 0.000 1.000
Dibenz(a,h)anthracene 0.930 1.13 1.461 0.144
pair of samples was not related (p < 0.01) to the mean concentra-
Indenopyrene 0.646 0.870 1.826 0.068
tions of that pair for any of the elements studied.
5506 A. Ares et al. / Atmospheric Environment 43 (2009) 5501–5509

5 10 15 20 25 30 35
10
10 15 20 25 30
Naphtalene Acenaphthene Fluorene Phenanthrene

8
Probability (X10-4)

Probability (X10-2)
Probability (X10-2)

Probability (X10-3)
8

6
6

4
020s
066m

4
082s
124s 105s
117s

2
133s 025s

2
5

B1 042s
B1 010s B3 A3 A2 A1
A1 B1 B1
B3
0

0
0
-400 -200 0 200 400 600 −10 0 10 20 30 40 −20 -10 0 10 20 30 0 100 200 300 400 500

7
10 15 20 25 30
Anthracene Fluoranthene Pyrene Benz(a)anthracene
25

25
A3

6
B4

Probability (X10-3)
Probability (X10-2)

Probability (X10-2)
Probability (X10-2)
20

20

5
117s
133s

4
15

15

010s
117s

3
10

10

129s 105s
042m 032s

2
B3 042s
5

1
A3 A2 B2 A1 A3 A3 A2 A1 025s 020s 042s
A2 A1 A2 A1
B1 B1

0
0
0

0
0 50 100 150 0 500 1000 1500 0 200 400 600 800 1000 0 50 100 150 200

20
8

Crysene Benzo(b+j)fluoranthene Benzo(k)fluoranthene Benzo(e)pyrene

8
8
Probability (X10-2)

Probability (X10-2)

Probability (X10-2)

Probability (X10-2)
15
6

6
6

10
4

4
4

5
2

2
2

B1 099s B3 B1
117m 129s A3 A2 A1 B4 B1 B3 B1
105s

0
0
0

−50 0 50 100 0 50 100 −10 0 10 20 30 40 −40 -20 0 20 40 60

15
8

Benzo(a)pyrene Benzo(ghi)perylene Dibenz(ah)anthracene Indenopyrene

Probability (X10-2)

8
Probability (X10-2)

Probability (X10-2)
Probability (X10-1)
15

10

6
10

4
5
5

2
082s A3 B3 A3 A1 B1 B3 B1 B3 B1
B1 A2
0
0
0
0

−10 0 10 20 -5 0 5 0 20 40 60 −20 0 20 40 60
Differences in concentration

Fig. 4. Kernel-smoothed estimates of the distributions of the differences in the concentrations of the 16 compounds determined in the moss Pseudoscleropodium purum between
pairs of sampling sites (SS) separated by approximately 1 km. The lines on the secondary x axis show the location of the values used to construct the distribution. For each
compound, the pairs of sampling sites affected by small scale contamination processes are indicated. For benzo(a)pyrene, the thick line shows the normal distribution calculated on
the basis of the mean values and standard deviations that are assumed not to be affected by sources of small scale contamination.

compounds. For the other compounds (i.e. anthracene, benz(a)an- 3.4. Application of the method to areas surrounding
thracene, crysene, fluoranthene and fluorene) there were several contamination foci
extreme values in the industrial areas and in the 2004 samples (see
Fig. 4). The pairs of SS from the 2004 samplings that were mainly The values of the differences in concentration for each
affected were: SS 042s, 105s and 117s. The SS pair 042s corresponded compound and each industry, along with the probability of
to extreme values of benz(a)anthracene, fluoranthene and fluorene; belonging to the distribution estimated in the previous section are
SS pair 105s to extreme values of crysene, fluoranthene and fluorene, shown in Table 3. Only the data corresponding to the radius of least
and SS pair 117s to extreme values of anthracene, fluoranthene and probability are shown (Table 2).
fluorene. The distribution of the mean values (and standard devia-
tions) of the differences in concentration between pairs of SS that were 4. Discussion
assumed not to be affected by small scale contamination are super-
imposed on the graph for benzo(a)pyrene, in Fig. 4. 4.1. Characterization of the PAH gradients in industrial areas
The resulting graph of the distribution of the differences in
concentration for all of the PAHs together is shown in Fig. 3, with The results confirm that the gradient of PAHs in moss samples
the different levels of significance for the differences between a pair collected in industrial areas is an exponential type, i.e. the
of SS considered as outside of the distribution shown on the graph. concentration of the contaminant is high in the immediate vicinity
The statistics and the concentrations corresponding to the of the source and decreases rapidly with increasing distance. This is
different quantiles of the distributions of the pairs of SS studied, for an essential condition for implementation of a method of identi-
each of the compounds and for the total PAHs are shown in Table 2. fying small scale contamination foci. Large differences in
 A. Ares et al. / Atmospheric Environment 43 (2009) 5501–5509 5507

Table 2 therefore the decrease in concentration was not as clear as for the
Parameters of the normal distributions of the differences in concentration -esti- other compounds (Fig. 2).
mated for each of the PAHs and for the sum of all the PAHs (ng g1) in the moss
Pseudoscleropodium purum – between pairs of sampling sites separated by approx-
The increase in concentration at point 4 in transect 1 at factory A
imately 1 km. The concentrations corresponding to various quantiles are shown. SD: may be due to the existence of an additional source of PAHs in this area.
standard deviation.

Mean SD Mode p  0.05 p  0.01 p  0.001 4.2. Differences between frozen samples and dried samples stored
Naphthalene 8.08 144 11.4 236 333 444 at room temperature
Acenaphthene 0.148 3.94 0.956 6.50 9.19 12.2
Fluorene 0.616 2.22 0.860 3.68 5.20 6.87
The results obtained in previous studies carried out with envi-
Phenanthrene 1.74 10.3 2.06 16.8 24.0 31.9
Anthracene 0.263 0.838 0.460 1.38 1.94 2.59 ronmental samples of a chemical matrix different from that of
Fluoranthene 2.10 4.70 0.156 7.60 10.9 14.5 terrestrial moss (Ambe, 1993; Kiriluk et al., 1996; Morita et al., 1997)
Pyrene 1.00 16.2 0.200 26.3 37.4 49.5 showed that for long term storage, freezing at 20  C appears
Benz(a)anthracene 1.02 8.80 0.117 14.5 20.6 27.3
adequate as this prevents any degradation by bacteria or enzymatic
Crysene 1.00 3.64 0.438 5.96 8.48 11.2
Benzo(b)fluoranthene 1.03 6.87 0.640 11.3 16.0 21.2
activity. However, in the present study, the results indicate that there
þ Benzo(j)fluoranthene was no significant degradation of PAHs in dried samples stored at
Benzo(k)fluoranthene 0.110 1.84 0.0374 3.03 4.28 5.66 room temperature. These findings may be of use to other researchers
Benzo(e)pyrene 0.860 4.95 0.279 8.20 11.6 15.3 as regards determining PAHs in the terrestrial moss P. purum if only
Benzo(a)pyrene 0.0166 4.20 0.238 6.90 9.79 13.0
this type of material (stored for up to four years) is available.
Benzo(ghi)perylene 0.0939 0.670 0.0636 1.11 1.56 2.08
Dibenz(a,h)anthracene 0.500 3.33 0.256 5.45 7.78 10.3
Indenopyrene 0.343 3.13 0.438 5.15 7.30 9.71
4.3. Distributions of the differences in concentration between the
SPAHs 17.4 232 18.6 384 540 717
pairs of sampling sites

The results obtained on establishing the distributions of the

concentrations of PAHs between the sample of moss collected from
close to the focus of contamination and the other collected at some
distance were observed, which confirms that the presumed source
was emitting the compounds. Only one previous study (Herrmann
and Hübner, 1984) has demonstrated this characteristic dispersion
of PAHs in relation to bioconcentration in native mosses; this was
proposed as one of the main objectives of the present study. The
gradient of decreasing concentration was observed for PAHs, both
at an individual level (Fig. 2) and for the sum of all of the PAHs
(Fig. 3). This is largely because PAHs are semivolatile compounds
and may show an affinity for binding to suspended particles
(particularly those compounds with 4 or more aromatic rings), so
that a sharp decrease was observed, particularly in comparison
with that of other contaminants such as PCBs, which have a higher
vapour pressure and for which the decrease in concentration is less
marked (Herrmann and Hübner, 1984). Naphthalene is the lightest
of the compounds studied, as it has only 2 aromatic rings, and
differences in concentration of the 35 pairs of SS indicate that,
except for benzo(a)pyrene, all compounds were normally distrib-
uted, with the mean equal to zero, after eliminating extreme values
that correspond to pairs of SS affected by small scale contamination
processes. The non-normal distribution of benzo(a)pyrene may be
explained by the fact that in some of the samples analysed, the
values obtained for this compound were below the limit of quan-
tification, and therefore on calculating the differences in concen-
tration, many of the values were close to zero, and the
corresponding distribution was leptocurtic.
In relation to the extreme values, most of the corresponding pairs
of SS were in the surroundings of the previously studied factories A
and B. The extreme values indicate the existence of contamination in
these industrial areas, which are important emitters of PAHs to the
atmosphere, from both the Fe–Mn smelter and the aluminium
smelter (Thomas and Schunke, 1984; Wegener et al., 1991).
For the other extreme values, corresponding to samplings carried
out within the 2004 Galician Air Quality Biomonitoring Network, it

Table 3
Differences between the values of the concentrations of the different PAHs (ng g1) in the moss Pseudoscleropodium purum corresponding to pairs of sampling sites in the
surroundings of different factories and the corresponding significance levels (p  0.05: italics; p  0.01: bold italics; and p  0.001: underlined bold). For Fe–Si smelter 3, T1
Indicates time of first sampling (2002), T2, indicates time of second sampling (2006). (Naphthalene: NAP, acenaphthene: ACE, fluorene: FLR, phenanthrene: PHE, anthracene:
ANT, fluoranthene: FLU, pyrene: PYR, benz(a)anthracene: BaA, crysene: CHR, benzo(b)fluoranthene þ benzo(j)fluoranthene: BbjF, benzo(k)fluoranthene: BkF, benzo(e)pyrene:
BeP, benzo(a)pyrene: BaP, benzo(ghi)perylene: BghiP, dibenz(ah)anthracene: DahA, indenopyrene: INP).

Factories NAP ACE FLR PHE ANT FLU PYR BaA CHR BbjF BkF BeP BaP BghiP DahA INP
Cement works 248 19.2 1.64 68.0 9.39 53.0 121 1.51 7.70 41.0 15.0 73.7 9.44 5.15 13.6 127
Ceramic factory 1 427 10.7 2.25 171 20.8 284 720 18.6 55.5 118 53.5 258 13.3 22.2 7.57 620
Ceramic factory 2 466 14.6 11.5 127 13.8 155 329 7.47 35.6 85.0 42.8 136 197 13.4 77.7 336
Ceramic factory 3 7.06 0.480 0.136 15.9 3.93 57.0 67.6 7.87 56.0 161 78.7 258 30.3 24.8 77.7 141
Glass factory 2.81 5.10 9.69 6.69 1.43 10.9 16.7 6.96 25.2 66.6 58.5 29.6 196 39.3 15.3 25.2
Fibreboard factory 1 139 6.60 7.37 20.4 3.89 20.0 41.4 6.30 15.3 30.3 23.0 33.6 3.96 6.36 12.0 31.0
Fibreboard factory 2 242 0.586 1.14 52.0 9.39 24.2 46.4 18.2 13.2 72.7 43.4 54.5 97.0 10.6 13.8 102
Fibreboard factory 3 146 10.2 16.6 84.0 8.19 170 220 12.8 39.0 78.0 51.0 63.0 6.50 9.89 37.9 145
Fe–Si smelter 1 114 14.6 3.75 89.0 15.4 222 185 97.9 99.9 258 161 177 110 38.9 98.9 173
Fe–Si smelter 2 389 33.6 23.0 74.7 4.18 108 84.0 23.4 12.8 104 36.0 50.5 18.8 6.78 52.5 64.0
Fe–Si smelter 3 (T1) 20.4 0.0900 3.20 26.8 1.83 33.0 38.9 78.7 448 999 616 846 262 55.0 37.6 101
Fe–Si smelter 3 (T2) 15.2 0.859 1.67 19.6 3.33 37.6 39.9 10.3 9.50 25.4 14.5 18.2 0.878 4.04 0.206 21.8
Aluminium smelter 18.2 7.37 9.29 474 144 1598 980 65.0 92.0 4.98 3.76 12.7 12.3 5.50 4.44 7.00
Biodiesel plant 196 0.970 1.30 44.0 8.50 57.5 98.9 0.0959 6.16 9.89 0.676 31.4 2.74 0.650 8.78 28.6
Waste treatment plant 35.6 9.99 4.74 143 13.2 181 343 1.45 44.8 53.0 16.5 128 16.2 7.57 57.5 284
Power station 382 6.36 3.03 123 15.6 121 240 0.570 25.2 40.4 4.54 202 4.94 16.0 13.3 276
5508 A. Ares et al. / Atmospheric Environment 43 (2009) 5501–5509
is not easy to provide a hypothesis regarding the focus of contami-
nation that affects these sites, as they are generally located within
forests or close to forests, and in most cases the sampling was carried
out in September. One possible explanation is that in the months
prior to sampling, the levels of hydrocarbons were higher in some SS
due to forest fires and/or agricultural activities during the summer.
The temporal variability in the structure that the foci generate must
be taken into account in developing the biomonitoring method. An
increase in temperature in summer may also increase the volatility
of the PAHs and greater amounts may therefore be registered on
some occasions in September.
On applying the method, certain problems that may arise should
be taken into account, as also pointed out by Fernández et al. (2007)
in the study concerning heavy metals and metalloids. Firstly, the
anisotropic effect, which is usually caused by the existence of pre-
vailing winds, as observed on studying the differences between the
two transects at factory B (Fig. 2). The prevailing winds probably blow
in the direction of transect 2, making the decrease in concentration
smoother in this direction and the area of influence of the focus of
contamination will be larger. Therefore because transect 1 was
almost perpendicular to transect 2, it probably reflected the gradient
of contamination better. The solution to this problem is therefore to
place two pairs of SS perpendicular to each other, with one orientated
in the same direction as the prevailing winds. Secondly, there
were problems related to the spatial structure around the focus of
contamination, as the two SS in each pair should be aligned in rela-
tion to each other and in relation to the focus of emission, as in
transect 1 at factory A, which is aligned with respect to the principal
source of emission from the factory (Fig. 1), focus ‘‘a’’, which is where
cathodes are elaborated (with coke as the fuel). Furthermore, the first
three SS in this transect were situated at increasing distances from
the source of contamination (1.I: 358m, 1.II: 421m and 1.III: 698m).
This should prevent the two SS in each pair forming a triangle with
the point of emission, as occurred in transect 2 at the same factory,
where the first three points were placed at approximately 1 km from
the source of emission (2.I. 937m, 2.II: 942m and 3.III. 1069m), and
thus the gradient of decrease in contamination was scarcely appre-
ciated. Another determining factor is the temporal variability that
may exist in the concentrations of PAHs in moss, due to environ-
mental conditions and/or the functioning of the focus of contami-
nation. Therefore in light of this possibility, the method should be
applied on more than one occasion if it is suspected that the
variability affects the contamination. Finally, one of the SS in the pair
should be located as close as possible to the focus of contamination to
detect the greatest differences in concentration.
4.4. Application of the method to areas surrounding
contamination foci
The results indicate that the industrial areas studied are sources of
small scale contamination by PAHs (Table 3), especially the heaviest
PAHs (i.e. crysene, benzo(b)fluoranthene þ benzo(j)fluoranthene,
benzo(k)fluoranthene, benzo(e)pyrene, benzo(a)pyrene, benzo(g-
hi)perylene, dibenz(ah)anthracene and indenopyrene). The latter
compounds may be deposited in greater amounts in the immediate
vicinity of the emission source, in contrast to other more volatile
compounds such as naphthalene, which may be transported longer
distances (Fig. 2).
In case of iron smelter 3, the probability associated with the
observed differences varied between the two sampling periods, so
that the concentrations of the compounds were much lower in T2
(second period) than in T1 (first period), especially for the heavier
compounds, as shown in Table 3. This indicates that the alterations
made (change of filters in the chimneys) were successful in
decreasing the contaminating compounds emitted.
Although benzo(a)pyrene was not normally distributed, the
quantiles calculated from the mean and the standard deviations of
the values of the differences in concentration between the pairs of
SS that were assumed not to be affected by small scale contami-
nation were applied. This may lead to an important loss of sensi-
tivity of the method, although it was sensitive enough to detect
small scale contamination foci, as shown in Table 3.
5. Conclusions
It was firstly demonstrated that, for the 16 compounds analysed
and for all compounds considered together, the gradient of decreasing
bioconcentration of PAHs in the moss P. purum followed an expo-
nential-type pattern. Secondly, sampling at a distance of 1 km is
optimal for the type of emission source studied. Thirdly, with the
exception of benzo(a)pyrene, the distribution of the differences in
concentration between the pairs of SS was normal, and extreme values
were observed in the immediate surroundings of diverse factories.
Despite certain problems that may arise during application of
the method, which may lead to loss of sensitivity, the results
indicate that the method was sufficiently sensitive to function
correctly for the detection of sources of small scale contamination
by PAHs with the terrestrial moss P. purum. In addition, the method
was found to be a very useful tool for monitoring PAHs and after
fine-tuning will be able to be used in other regions and with other
types of emission sources.
Acknowledgements
The present study was partially funded by the Xunta de Galicia
as part of the project entitled: ‘‘Apoyo a la implementación de la Ley
16/2002 de prevención y control integrados de la contaminación’’. We
thank Cristina Gianzo and Mercedes Noya for processing the moss
samples and also Dr Purificación López and Dr Marı́a Piñeiro from
the Instituto de Medio Ambiente at the University of La Coruña for
carrying out the chemical analyses.
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