Urban Forestry & Urban Greening 25 (2017) 1–10

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Urban Forestry & Urban Greening

journal homepage: www.elsevier.com/locate/ufug

Urban background of air pollution: Evaluation through moss bag MARK

biomonitoring of trace elements in Botanical garden
⁎
Mira Aničić Uroševića, , Gordana Vukovića, Petar Jovanovićb, Milorad Vujičićc,
Aneta Sabovljevićc, Marko Sabovljevićc, Milica Tomaševića
a
Institute of Physics Belgrade, University of Belgrade, Pregrevica 118, 11080 Belgrade, Serbia
b
Institute for Multidisciplinary Research, University of Belgrade, Kneza Višeslava 1, 11030 Belgrade, Serbia
c
Institute of Botany and Garden, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia

A R T I C L E I N F O A B S T R A C T

Keywords: Urban background air pollution is the lowest level of pollution representative for the exposure of general urban
H. cupressiforme population and mainly originates from non-local sources of pollution. Moss bag technique has been
Moss bag biomonitoring predominantly adopted for biomonitoring of trace elements across anthropogenically devastated areas, such
Pigments as urban and industrial zones. However, the technique has been rarely used for measurement of background air
Phenolic content
pollution. In this study, element content and concomitant physiological parameters were assessed in the moss
Trace elements
Hypnum cupressiforme Hedw. after its exposure within the bags in the Botanical garden (Belgrade, Serbia), as the
presumable background area. During the summer of 2014, the moss bags were exposed for 60 days in total, and
the sample analyses were performed every 15 days. As a control of the measured physiological parameters, a set
of the moss bags was kept within the phytotron, under conditions of optimal-like growth. The total content of 21
elements, photosynthetic pigments (chlorophylls and carotenoids), phenolics, and antioxidative capacity were
determined in the moss samples. The levels of trace elements were markedly lower within the Botanical garden
in comparison to the street ambient, but still significantly higher than in the moss pristine habitat. However, the
performed physiological tests indicated that the vulnerability of the moss during exposure time was not caused
by the low trace element enrichment, but probably by the harsh meteorological parameters in an urban area.
Finally, the Botanical garden could be assumed as control site for measurement of the urban background
pollution, and it might be effectively measured by the moss bag technique.

1. Introduction
Over 70% of the human population across Europe lives in urban
agglomerations and, to some extent, suffers from influence of air
pollution (EEA, 2014). Field measurements highlight a complexity of
pollutant emissions, as well as their transport and dispersion across the
urban settings (Baldauf et al., 2013). Topography and urbanisation
features of the cities cause high spatial variability of air pollutant
concentrations.
The total concentration of pollutants comprises those from the
explicit local emission sources such as road traffic and chimney stacks;
and those called ‘background’ air pollution that is transported into an
area from distant sources by the wind (Hertel and Goodsite, 2009;
DEFRA, 2014). Considering the regulatory documents, ‘urban back-
ground locations’ mean places in urban areas where pollutant levels are
representative of the exposure level of general urban population
(Directive 2008/50/EC). In reality, measurements of urban air pollu-
tion are largely available for non-representative sampling sites within
the urban area. Air pollution monitoring stations are usually situated at
presumably pollution ‘hot spots’, and rarely in areas with a lower level
of air pollution. Thus, data about urban background pollution level are
not practically available. Measurements of this kind lack worldwide and
these might be only estimated using statistical models (Cohen et al.,
2004).
Biomonitoring is a simple method for estimating the pattern of
atmospheric pollution in a certain area using living systems, such as
mosses or lichens (Markert et al., 2003; Pirintsos and Loppi, 2008;
Aničić Urošević et al., 2017). Particularly, active biomonitoring refers
to the use of suitable moss or lichen species transplanted from
unpolluted area to the sites of interest. This approach enables inte-
grated monitoring of many airborne contaminants simultaneously (i.e.,
metals and metalloids, trace elements, polycyclic aromatic hydrocar-
bons – PAHs, radionuclides) (Giordano et al., 2005; Ares et al., 2012;
Vingiani et al., 2015; Vuković et al., 2015; Krmar et al., 2016). It is hard

⁎
Corresponding author at: Environmental Physics Laboratory, Institute of Physics Belgrade, University of Belgrade, Serbia.
E-mail address: mira.anicic@ipb.ac.rs (M.A. Urošević).

http://dx.doi.org/10.1016/j.ufug.2017.04.016
Received 17 October 2016; Received in revised form 24 April 2017; Accepted 27 April 2017
Available online 30 April 2017
1618-8667/ © 2017 Elsevier GmbH. All rights reserved.
M.A. Urošević et al.
for any other monitoring approach to enable such a detailed picture of
the pollutant variations in space, and at a reasonable cost. The
advantages of biomonitor use over regular monitoring are the simpli-
city of performing and the lack of the need for power supply.
‘Moss bag technique’ has been developing in last several decades as
a method for active biomonitoring of air pollutants (Ares et al., 2012).
This method has been particularly useful for conducting a detailed
survey in diversely polluted urban microenvironments where native
mosses are usually absent because of predominantly paved and land-
scaped surfaces. Specifically, the moss collected from relatively un-
polluted area, with known initial element content, could be exposed in
the bag at the site of interest for a certain period acting as an integrated
accumulator of the ambient element pollution expressed by net
concentrations or relative accumulation factor. However, relocation
of moss from their natural habitat for the purpose of active biomonitor-
ing represents a substantial stress for the moss due to changed macro-
and microclimate and meteorological conditions. The exposure in the
suspended bags is probably a drastic change of moss natural habitat
predominantly due to hydric and photo stress (Tretiach et al., 2007).
Moreover, in the conditions of air pollution burden, such as in urban
area, the physiological parameters of the moss could reflect the heavy
metal pollution, especially when ambient pollution is continuous for
several weeks or months (Sun et al., 2009).
In this study, active moss biomonitoring was conducted within the
Botanical garden with the aim to assess whether the gardens situated in
the downtown area could be assumed as a place for the assessment of
urban background pollution. In addition, it was investigated whether
presumably low trace element load in the Hypnum cupressiforme moss
bags causes disorder of physiological state of the exposed moss, which
implies active or passive element uptake.
2. Materials and methods
2.1. Study area
The study area was Botanical garden “Jevremovac” within Belgrade
downtown (Serbia) (44°48′56.9″N; 20°28′23.6″E). The arboretum
spreads over the area of 50,000 m2 of open space and includes over
300 tree species. The Botanical garden is bounded with two streets with
very frequent traffic, Boulevard of Despot Stefan and Takovska St., and
by the three less car-occupied streets (Fig. 1).
Belgrade has a moderate continental climate. The year-round
average temperature is 11.7 °C, the hottest month is July, with an
average temperature of 22.1 °C; annual precipitation is about 700 mm.
During the study period (July–September 2014), an average tempera-
ture was 20.8 °C while the precipitation varied from 8 mm to150 mm
with an average value of 48.7 mm (Table 1, Supplementary material).
2.2. Moss sampling and bag preparation
In May of 2014, the moss H. cupressiforme Hedw. was collected in
the protected area “Vršačke planine’, Serbia (44°08′26″N; 21°23′43″E;
230 m a.s.l.) that is recognised as an appropriate pristine area for the
moss sampling (Vuković et al., 2015, 2016). The moss was sampled on
the rules of the international moss monitoring manual (ICP Vegetation,
2005). In the laboratory, green apical parts of the moss were manually
cleaned and separated from the rest of the plant. Subsequently, the
selected green parts of the moss were rinsed three times with double-
distilled water (approximately 10 L water per 100 g of moss dry weight
and 10 min of shaking). The moss was air-dried and gently hand-mixed.
Approximately 1.5 g of the moss was packed loosely in 7 × 7 cm nylon
net bags with a 2-mm mesh size resulting in a surface area of
approximately 30 mg cm−2, which is suggested by Ares et al. (2012)
to be the most suitable for maximum uptake (Aničić et al., 2009a;
Vuković et al., 2015). The nylon is inert material and does not interfere
with the element uptake process while mesh has a homogenising effect
2
Urban Forestry & Urban Greening 25 (2017) 1–10
on element entrapment acting as a sieve for large airborne particulates
(Ares at al., 2012). Moreover, moss material was packed as thin as
possible to avoid its overlapping.
To avoid contamination of the moss material, the entire process of
moss sampling and bag preparation was conducted wearing polyethy-
lene powder-free gloves. Several moss bags were stored at room
temperature in the laboratory conditions for determination of the
initial pollutant concentrations in unexposed moss material.
2.3. Experimental design
The study was carried out in the Botanical garden from July to
September 2014. The moss bags were exposed to atmospheric deposi-
tion for four consecutive exposure periods – 15, 30, 45, and 60 days at
two sites – garden boundary and garden inner (Fig. 1). Ten moss bags
per study site (four for chemical analysis, and four for physiological
tests; two moss bags were extra added for the case of bag loss from any
reasons) were hung on plasticized aluminium holders specifically
designed for that purpose. The holders were mounted perpendicular
to the lampposts at the representative height of 3.5 m (Vuković et al.,
2013) in open spaces, out from tree canopies and electric cables. As
control samples for the atmospheric deposition of trace elements in
open spaces, ten moss bags were sheltered under the roof, close to
garden boundary site, and wetted several times per week by spray-
distilled water. An aliquot of unexposed moss collected in the same
pristine area was used to measure the pre-exposure elemental concen-
trations (in triplicates).
2.4. Chemical analysis
After each exposure periods, one moss bag was moved per studied
site, and further, the moss material were air-dried and homogenised in
the laboratory conditions. Each moss sample was analysed in triplicates
(n = 3 subsamples × 4 exposure periods × 3 study sites = 36).
Approximately 0.3 g of each moss subsample were dissolved with
1 mL of 30% H2O2 (Sigma-Aldrich, puriss. p.a.) and 7 mL of 65%
HNO3 (Sigma-Aldrich, puriss. p.a., distilled by the apparatus for acid
distillation – BERGHOF, Products + Instruments GmbH, Germany).
Further digestion procedure was performed in a microwave digester
(ETHOS 1, Advanced Microwave Digestion System, Milestone, Italy) for
45 min at 200°C. The digested samples were diluted with double-
distilled water to a total volume of 50 mL.
The concentrations of Al, Ba, Cu, Fe, K, Mg, Mn, Na and Zn were
determined using inductively coupled plasma-optical emission spectro-
scopy (ICP-OES, Thermo Scientific iCAP 6500 Duo, Thermo Scientific,
UK). For calibration, a Multi-Element Plasma Standard Solution 4,
Specpure (Alfa Aesar GmbH & Co KG, Germany) was used. The con-
centrations of As, Cd, Ce, Co, Cr, Ni, Pb, Rb, Sb, Sn, Sr and V were
determined using inductively coupled plasma-mass spectrometry (ICP-
MS, Thermo Scientific iCAP Q, Thermo Scientific, UK). A low-level
Elements Calibration Stock, EPA Method Standard (VHG Labs,
Manchester) was used for calibration.
Quality control of the element determination was provided through
analysis of the analytical blanks and certified reference materials that
were analysed once every ten moss samples. As the certified reference
materials, the moss Pleurozium schreberi M2 and M3 (Steinnes et al.,
1997) were used. The recovery of elements from the reference material
M2 ranged from 75% to 111% for the majority of the determined
elements; however, the recoveries of Ni and Ce were 126% and 56%,
respectively. For the reference material M3, the recovery of elements
ranged from 80% to 112%, whereas those of Cu and Ce were 63% and
57%, respectively. The data were corrected depending of the element
concentration level by the mean recovery value (%) either M2 or M3.
M.A. Urošević et al. Urban Forestry & Urban Greening 25 (2017) 1–10

Fig. 1. Map of the study area. The Botanical garden “Jevremovac”: study sites – garden boundary and garden inner.

Table 1
The values of meteorological parameters during the experimental set up (July–September, 2014).

Month, 2014 Т average (оС) Т max (оС) Т min (оС) Insolation (h) Precipitation (mm)

21–31 July 23.3 > 30 (30.6/1 day) 17.7 71 150

1–10 Aug 23.5 > 30 (31.3/2 days) 17.9 67 33
11–20 Aug 24.1 > 30 (34.8/4 days) 13.9 101 8
21–31 Aug 21.1 29.4 11.9 95 22
1–10 Sep 21.1 28.2 15.7 34 46
11–20 Sep 20.4 28.3 14.3 53.7 47.8
21–30 Sep 16.1 25.5 8 77 34

2.5. Physiological tests
After each exposure period, the pigments (chlorophylls and carote-
noids), total phenolics and antioxidative capacity were determined in
the moss bag sample, in triplicates (n = 3 subsamples × 4 exposure
periods × 4 study sites = 48). As the control samples for physiological
tests, the moss was kept hydrated and placed in a phytotron under the
controlled light (16 h light and 8 h night) and temperature (18 ± 2°C)
conditions.
Photosynthetic pigments were extracted from 100 mg frozen moss
material in 96% (v:v) ethanol and the absorbance of ethanol extract was
measured with UV–vis Spectrophotometer (HP/Agilent 8453
Spektrofotometеr, Agilent technologies, Paolo Alto, CA) at three
wavelengths: 470 nm, 648 nm, and 664 nm (Lichtenthaler, 1987).
Total phenolic content (TPS) was analysed using the modified Folin-
Ciocalteu method (Dewanto et al., 2002). Polyphenols were extracted
by adding 2 mL of 96% ethanol to 200 mg of the moss tissue. To 60 μL
of the extract, 1340 μL of sterile distilled water, 300 μL of Folin-
Ciocalteu reagent, and 300 μL of 20% Na2CO3 were added. The mixture
was standing at the room temperature in dark for 1.5–2 h, and the
absorbance of samples was measured at 765 nm. Gallic acid was used
for constructing a calibration curve. Results were expressed as mg of
gallic acid per 100 g of the moss fresh weight.
Antioxidative activity of the fresh extract was measured by the
scavenging of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical at
520 nm. Further, 0.15 mM DPPH was prepared in methanol. The
reaction mixture was comprised of DPPH solution 0.4 mL, 0.5 mL of
methanol and 0.1 mL of the sample and allowed to stand for 45 min and
its absorbance was measured at 520 nm (Brand-Williams et al., 1995).
Antioxidative activity of the moss materials was expressed as the
percentage of free radical scavenging.
2.6. Data processing
To assess the element accumulation of the studied moss species,
relative accumulation factor (RAF) was calculated as the moss content
of each element after exposure (Cexposed) subtracted and then divided by
the element content before exposure (Cinitial):
RAF = (Cexposed − Cinitial)/Cinitial.
The amount of pigments, chlorophylls and carotenoids (mg g−1 of
dry weight), were calculated using the following formulas
(Lichtenthaler, 1987):
Chl(a+b) = 5.24 × A664 + 22.24 × A648
Chla = 13.36 × A664 − 5.19 × A648
Chlb = 27.43 × A648 − 8.12 × A664
C(x+c) = 1000 × A470 − 2.13 Chla − 97.64 Chlb/209,
where are: Chla − Chlorophyll a; Chlb − Chlorophyll b; Ca+b − Total
chlorophylls; Cx+c − Total carotenoids; A648 = Absorbance at 648 nm;
A664 = Absorbance at 664 nm; and A470 = Absorbance at 470 nm.

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M.A. Urošević et al. Urban Forestry & Urban Greening 25 (2017) 1–10

Table 2
Descriptive statistics and RAF values for the element concentrations (μg g−1 dw) determined in the moss H. cupressiforme after different exposure periods (15–30–45–60 days) at: garden
boundary, garden inner, control-sheltered site; initial – unexposed moss material, RAF – relative accumulation factor, Mean – average values, SD – standard deviation.

Element 0 15 days 30 days 45 days 60 days

Initial Control- Garden Garden Control- Garden Garden Control- Garden Garden Control- Garden Garden
sheltered boundary inner sheltered boundary inner sheltered boundary inner sheltered boundary inner

Al Mean 735 1002 786 837 1030 1436 1026 998 1443 990 1162 868 1334
RAF 0.4 0.1 0.1 0.4 1.0 0.4 0.4 1.0 0.3 0.6 0.2 0.8
SD 151 45 86 62 71 598 212 215 48 21 272 74 412
As Mean 0.345 0.417 0.343 0.337 0.455 0.598 0.407 0.449 0.592 0.418 0.456 0.335 0.536
RAF 0.21 −0.01 −0.02 0.32 0.73 0.18 0.30 0.72 0.21 0.32 −0.03 0.55
SD 0.062 0.023 0.024 0.064 0.041 0.252 0.092 0.057 0.049 0.020 0.137 0.061 0.127
Ba Mean 16.2 18.4 16.9 17.5 17.5 17.3 17.3 20.0 17.7 17.4 16.7 16.3 17.3
RAF 0.14 0.04 0.08 0.08 0.07 0.07 0.24 0.09 0.07 0.03 0.01 0.07
SD 0.5 1.1 2.0 0.3 1.4 2.1 1.0 0.6 0.3 1.3 1.4 1.6 1.4
Ca Mean 5633 5532 5205 5991 5573 5174 5798 6262 5218 5457 5254 5347 5154
RAF −0.02 −0.08 0.06 −0.01 −0.08 0.03 0.11 −0.07 −0.03 −0.07 −0.05 −0.09
SD 20 379 649 295 281 209 484 162 33 162 465 278 160
Cd Mean 0.323 0.451 0.407 0.383 0.420 0.412 0.351 0.429 0.394 0.402 0.378 0.294 0.397
RAF 0.39 0.26 0.19 0.30 0.28 0.09 0.33 0.22 0.24 0.17 −0.09 0.23
SD 0.008 0.048 0.164 0.037 0.011 0.023 0.051 0.020 0.017 0.034 0.017 0.035 0.032
Ce Mean 1.08 1.36 0.99 1.15 1.46 1.94 1.33 1.50 1.87 1.21 1.47 1.03 1.81
RAF 0.26 −0.09 0.06 0.34 0.79 0.23 0.38 0.73 0.12 0.36 −0.05 0.67
SD 0.16 0.06 0.11 0.13 0.22 0.84 0.30 0.28 0.08 0.01 0.44 0.15 0.46
Co Mean 0.375 0.422 0.369 0.370 0.479 0.527 0.417 0.458 0.438 0.388 0.418 0.356 0.503
RAF 0.13 −0.01 −0.01 0.28 0.41 0.11 0.22 0.17 0.04 0.12 −0.05 0.34
SD 0.023 0.009 0.015 0.009 0.051 0.076 0.069 0.029 0.015 0.009 0.045 0.024 0.076
Cr Mean 0.83 0.96 0.86 0.94 1.12 1.38 1.10 1.12 1.17 1.03 1.14 1.06 1.32
RAF 0.2 0.0 0.1 0.4 0.7 0.3 0.3 0.4 0.2 0.4 0.3 0.6
SD 0.12 0.03 0.15 0.07 0.09 0.34 0.18 0.18 0.07 0.01 0.15 0.06 0.28
Cu Mean 8.0 8.7 7.2 8.7 8.8 8.9 8.7 9.3 9.0 9.0 8.5 8.7 9.4
RAF 0.08 −0.09 0.08 0.10 0.12 0.09 0.16 0.12 0.13 0.07 0.09 0.18
SD 0.1 0.4 1.1 0.5 0.3 0.4 0.2 0.3 0.3 0.5 0.0 0.4 1.0
Fe Mean 645 805 673 696 836 1074 786 806 1071 809 906 752 1040
RAF 0.25 0.04 0.08 0.29 0.66 0.22 0.25 0.66 0.25 0.40 0.17 0.61
SD 125 30 56 26 64 342 142 96 45 14 150 46 243
K Mean 4977 4989 4523 4503 4997 5191 5129 4745 5166 5044 4920 4431 4549
RAF 0.002 −0.091 −0.095 0.004 0.043 0.031 −0.047 0.038 0.013 −0.011 −0.110 −0.086
SD 45 118 135 21 30 151 130 195 185 116 87 308 73
Mg Mean 1670 1404 1356 1488 1402 1436 1463 1531 1413 1434 1439 1395 1322
RAF −0.16 −0.19 −0.11 −0.16 −0.14 −0.12 −0.08 −0.15 −0.14 −0.14 −0.16 −0.21
SD 7 68 125 4 50 96 63 17 8 40 71 83 60
Mn Mean 109 107 102 116 112 113 114 130 114 105 110 99 108
RAF −0.01 −0.06 0.07 0.03 0.04 0.05 0.20 0.04 −0.03 0.01 −0.09 0.00
SD 3 4 9 3 8 7 6 0 7 3 16 13 5
Na Mean 35.0 28.1 15.8 19.3 38.9 40.8 43.3 35.1 47.9 43.1 35.4 32.2 32.8
RAF −0.20 −0.55 −0.45 0.11 0.17 0.24 0.01 0.37 0.23 0.01 −0.08 −0.06
SD 1.9 2.3 0.1 4.2 10.9 7.5 5.5 8.5 5.3 3.7 10.0 3.9 9.8
Ni Mean 1.54 1.50 1.36 1.45 2.21 1.71 1.56 1.59 1.79 2.16 1.56 1.57 1.77
RAF −0.03 −0.12 −0.05 0.44 0.11 0.02 0.03 0.17 0.41 0.01 0.02 0.15
SD 0.67 0.05 0.13 0.19 0.53 0.21 0.19 0.11 0.29 0.05 0.08 0.18 0.24
Pb Mean 6.2 10.8 7.2 9.3 9.0 8.5 7.1 10.6 9.8 9.3 8.5 6.0 9.3
RAF 0.74 0.16 0.50 0.45 0.37 0.15 0.70 0.57 0.50 0.36 −0.03 0.50
SD 0.6 1.0 2.1 0.5 0.9 1.4 1.6 0.3 0.3 1.1 0.2 0.9 1.4
Rb Mean 5.54 6.38 5.08 5.70 6.38 7.04 6.47 6.47 7.35 6.30 6.16 5.11 6.38
RAF 0.15 −0.08 0.03 0.15 0.27 0.17 0.17 0.33 0.14 0.11 −0.08 0.15
SD 0.14 0.13 0.81 0.26 0.09 1.17 0.38 0.21 0.11 0.20 0.44 0.62 0.80
Sb Mean 0.034 0.049 0.072 0.056 0.050 0.056 0.061 0.061 0.060 0.080 0.068 0.092 0.077
RAF 0.4 1.1 0.7 0.5 0.7 0.8 0.8 0.8 1.4 1.0 1.7 1.3
SD 0.009 0.005 0.020 0.007 0.005 0.002 0.004 0.011 0.014 0.020 0.018 0.027 0.009
Sn Mean 0.016 0.016 0.016 0.012 0.015 0.022 0.029 0.011 0.033 0.023 0.018 0.019 0.019
RAF 0.02 −0.03 −0.26 −0.05 0.38 0.79 −0.34 1.07 0.43 0.12 0.20 0.21
SD 0.020 0.012 0.009 0.011 0.006 0.018 0.006 0.017 0.003 0.011 0.018 0.015 0.027
Sr Mean 18.0 19.0 16.6 20.2 18.5 17.7 20.2 22.3 19.0 19.8 17.5 18.0 17.7
RAF 0.1 −0.1 0.1 0.0 0.0 0.1 0.2 0.1 0.1 0.0 0.0 0.0
SD 0.3 1.0 5.3 1.9 0.8 1.8 1.6 1.1 0.2 1.2 2.2 1.5 1.9
V Mean 1.31 1.43 1.29 1.43 1.74 2.04 1.66 1.68 1.79 1.59 1.70 1.58 2.07
RAF 0.091 −0.011 0.093 0.330 0.563 0.271 0.287 0.368 0.217 0.302 0.210 0.584
SD 0.16 0.07 0.12 0.04 0.07 0.50 0.27 0.23 0.12 0.03 0.22 0.09 0.40
Zn Mean 28.7 40.8 55.3 46.9 44.8 37.5 44.2 40.4 44.0 39.7 34.3 81.7 166.4
RAF 0.4 0.9 0.6 0.6 0.3 0.5 0.4 0.5 0.4 0.2 1.8 4.8
SD 0.7 4.7 27.4 13.4 9.9 2.6 11.4 2.7 10.4 2.3 2.0 10.7 138.5

4
M.A. Urošević et al. Urban Forestry & Urban Greening 25 (2017) 1–10

Fig. 2. Average concentration (μg g−1 dw) of Sb in the moss H. cupressiforme exposed at three sites within the Botanical garden during the summer of 2014; initial – Sb concentration in
the unexposed moss.

The data were processed using STATISTICA 8.0 (StatSoft Inc., Tulsa, street (Fig. 2). According to the field and laboratory studies, Sb may
OK, USA). The normality of the data was tested by Shapiro–Wilk test greatly accumulate in biomonitor such as lichens (Paoli et al., 2013a,
(p < 0.05). For testing differences in obtained physiological para- 2013b), and it originates from a vehicle traffic even in a low polluted
meters, Fridman‘s two-way ANOVA was applied at significance level environment such as botanical garden (Loppi and Paoli, 2015). Thus,
p < 0.05. To determine significant differences (p < 0.05) in the based on the results of this study, the ‘optimal’ bag exposure time could
elemental concentrations between different periods of the moss ex- not be recommended for assessment of urban background pollution.
posure, and different study sites, Mann–Whitney U test was used. The ‘optimal’ time refers to the minimal period for achieving the
significant and consistent element uptake that reflects the ambient
element content.
3. Results and discussion
Finally, the element enrichment in the moss samples was low
probably due to low air pollution burden within the Botanical garden,
3.1. Moss element content vs. exposure time
which is covered with vegetation and surrounded by a hedge (trees/
bushes), i.e., vegetation barrier against pollution. However, 60-day
The content of elements in the moss samples is the result of gradual
moss exposure period was selected for further comparison since it had
uptake, but also of the loss of some elements by leaching. Therefore, the
been practised as ‘optimal’ in the previous studies performed in the
element content in the samples cannot exhibit linear relationships with
Belgrade urban area (e.g., Aničić et al., 2009b; Vuković et al., 2016). In
levels of atmospheric deposition and mostly depends on bag exposure
this study, the moss element concentrations were significantly lower
time (Ares at al., 2012). In this study, the moss was exposed for
(p < 0.05) than in the street biomonitoring study conducted during
15–30–45–60 days to discover the ‘optimal’ bag exposure time in urban
the same summer season (Vuković et al., 2016) where the H. cupressi-
ambient such as a botanical garden. After the exposure, the concentra-
forme moss bags were exposed at the crossroads and two- and one-lane
tions of Ca, K and Mg were even lower than in the initial moss tissue,
streets close to the Botanical garden.
which is also observed in the previous studies (Aničić et al., 2009a,b;
Calculation of the element RAFs is a good way to eliminate the
Vuković et al., 2013). The moss concentrations of Al, Cr, Cu, Fe, Pb, Sb,
initial element content in the exposed moss and to make intra/inter-
V, and Zn were significantly higher (p < 0.05) at the garden inner and
study comparisons. Since the element enrichment in the moss was
boundary sites, and even the control-sheltered site (Table 2, Supple-
similar through the periods of bag exposure (15–30–45–60), mean RAFs
mentary material). However, the element concentrations did not
were calculated for all periods. The highest RAFs were calculated in the
significantly differ between the moss exposed at the control-sheltered
moss exposed close to the boundary of the Botanical garden, influenced
site and the garden boundary site in open space.
by the highest traffic flow impact of the neighbouring streets. However,
Although the element concentrations were gradually (slightly)
only RAF values of Sb, Cr, Al, and V were significantly higher
increasing with duration of the exposure periods, the element concen-
(p < 0.05) in the moss exposed at the garden boundary site compared
trations did not significantly differ (p < 0.05) in the moss bags
to the inner exposure site. Mean RAFs decreased from the garden
between 15 and 60 days, except for Sb. Antimony as a key tracer of
boundary to the inner and following the sheltered site (Fig. 3a).
non-exhaust traffic, i.e., deterioration of tire, brake, engine and vehicle
Calculated RAF values for the elements in this study were markedly
component (Fujiwara et al., 2011; Grigoratos and Martini, 2015
lower than those obtained in the comparative studies carried out in the
Grigoratos and Martini, 2015), was the most enriched element in the
typical urban microenvironments, close to the streets/crossroads in the
moss samples (Table 2, Supplementary material), especially at the
Belgrade urban area (Vuković et al., 2015, 2016) (Fig. 3b). The element
garden boundary site, which is relatively close to the frequent traffic

5
M.A. Urošević et al.
Fig. 3. Mean values of relative accumulation factors (RAFs) for the element content in the H. cupressiforme moss bags exposed during the summer of 2014 at: a) three sites within the
Botanical garden; dotted lines represent significant (p < 0.05) trends; and b) the Botanical garden, and street/crossroad sites of the comparable street study (Vuković et al., 2016).
RAF values calculated for the H. cupressiforme moss bags exposed at the
neighbouring crossroad were 10–90 times higher than in the Botanical
garden, especially for Ba, Cr, Cu, Sb, Sn, V, and Zn. In urban areas, these
elements are usually the product of exhaust and non-exhaust traffic
emissions (Pant and Harrison, 2013). Among the studied elements
within the Botanical garden, the moss entrapment of Sb was the highest
in comparison to its initial value, such as in the street study (Fig. 3b)
(Vuković et al., 2016). Besides vehicle-specific brake wear emissions,
road dust resuspension was a dominant contributor of Sb in the streets
(Bukowiecki et al., 2009).
3.2. Eco-physiological state of moss vs. element moss enrichment
Moss H. cupressiforme is a ubiquitous, light and drought tolerant
6
Urban Forestry & Urban Greening 25 (2017) 1–10
species. Nevertheless, relocation of moss from their natural habitat in
the suspended bags represents a drastic change of the moss environ-
ment. Pre-treatment of the moss in the process of the bag preparation
and its later exposure within the polluted urban area can be considered
as substantial stress for the moss tissue. In this study, even the first 15-
day exposure significantly (p < 0.05) reduced the vitality of moss in
bags. This phenomenon was proved by the changes of photosynthetic
pigments (Fig. 4).
Specifically, after 15 days, photosynthetic pigments were consider-
ably lower in the moss, even in those exposed in phytotron: the content
of total chlorophylls and carotenoids decreased by approximately 20%
and 30% of their initial value, respectively. However, after 45 days, the
pigment content was increasing in the control moss exposed in the
optimal-like pre-defined condition of phytotron.
M.A. Urošević et al.
Fig. 4. The content of total chlorophylls and carotenoids (mg g−1 dw) in the moss H. cupressiforme exposed within the Botanical garden at three sites, and in phytotron during the summer
of 2014; initial – the pigment content in the unexposed moss.
As widely reported in the literature, the concentrations of photo-
synthetic pigments may indicate physiological disorders of moss during
senescence or stress (Aboal et al., 2008; Niinemets and Tobias, 2013).
In the conditions of highly-polluted urban area, burden by heavy metals
and another toxic element could be assumed as eco-physiological stress
for moss biomonitors that cause chlorophyll loss. This was noticed by
treating mosses with different concentrations of Cu, Pb and Zn in the
laboratory conditions (Shakya et al., 2008). In addition, the moss
exposed in bags in urban environment lose viability in a short time
(Tretiach et al., 2007; Spagnuolo et al., 2011) and the moss passively
entraps airborne elements as PM which remain on its surface. However,
Tremper et al. (2004) did not observe any decrease in chlorophyll
concentration depending on metal pollution after moss exposure in
semi-urban and beside-road zones. The authors suggested that other
7
Urban Forestry & Urban Greening 25 (2017) 1–10
factors such as climatic factors and interactions with other airborne
contaminants would probably have a greater influence on the chlor-
ophyll concentration. Furthermore, Varela et al. (2013) pointed out that
the level of chlorophyll in the transplanted moss would not decrease
until the threshold concentration of metal in the moss tissue is reached.
Finally, different environmental factors may influence variations in the
pigment contents, and such analysis is not specific to a certain kind of
stress, but rather depicturing a synergy of various environmental
factors.
In this study, the antioxidant activities of the moss were also
investigated regarding TPC and DPPH radical scavenging assays
(Figs. 5 and 6, respectively). TPC and DPPH activity were several times
lower in the control moss kept in a phytotron than in the moss exposed
in bags at the study sites. The hydration of the moss plays a significant
M.A. Urošević et al.
Fig. 5. Total phenolic concentration (mg GA g−1 dw) in the moss H. cupressiforme exposed during the summer of 2014 within the Botanical garden at three sites, and in phytotron; and in
the unexposed moss (initial).
Fig. 6. DPHH activity (% dw) in the moss H. cupressiforme exposed during the summer of 2014 within the Botanical garden at three sites, and in phytotron; and in the unexposed moss
(initial).
role in their vitality. Thus, the prominent rainy period from 30th to
45th day of the moss exposure (Table 1, Supplementary material)
caused a significant decrease (p < 0.05) of TPC and DPPH activity.
Moreover, TPC was rather lower in the sheltered (in shadow) moss,
especially after 60 days. This result confirms findings that phenolics
play a role in photoprotection (Dey and De, 2012).
The moss exposed in bags suffers partly due to suboptimal eco-
physiological conditions in the urban area, but also due to the uptake of
air pollutants. On one hand, heavy metal-induced toxicity in moss
includes alteration of enzyme activities, damage to chlorophyll bio-
synthesis and content, and other physio-morphological characteristics.
On the other hand, moss species that thrive in metal-rich habitat
become tolerant due to hyperaccumulation of metals or sequestering of
excessive metal ions without disturbing metabolic activities (Dey and
8
Urban Forestry & Urban Greening 25 (2017) 1–10
De, 2012). In this study, the low element content obtained in the
exposed moss bags suggests a relatively low level of air pollution in the
Botanical garden by the elements in question. In these conditions, the
measured element load probably could not affect the changes of eco-
physiological parameters in the exposed moss. The changing of the
pigment content and antioxidative capacity of the moss was rather
caused by suboptimal meteorological conditions in the study area in
comparison with the pristine area or phytotron. Except for the
phytotron-moss, the majority of the tested eco-physiological parameters
did not significantly differ (p < 0.05) among the moss bags at three
other sites – garden boundary, sheltered and garden inner. This result
could be a consequence of relatively homogeneous environmental
conditions (e.g., temperature, humidity) within the Botanical garden
that could represent key factors for maintaining stable moss eco-
M.A. Urošević et al.
physiological parameters. Thus, in biomonitoring studies, the moss bag
entrapment of trace elements should be also compared to optimal and
suboptimal conditions, and surely will be different in areas with a
different climate, or during different seasons.
3.3. Could botanical garden be considered as an urban background?
Without local pollution sources, all that remains in a certain area is
‘background’ pollution, which comes from distant sources. The mea-
surements of this level of air pollution could be of importance to
provide estimates of background concentrations for specific pollutants
(DEFRA, 2014). Although the studied Botanical garden is situated in the
very central area of Belgrade and surrounded by heavily loaded streets,
the garden could be assumed as less air polluted ‘niche’ within the
considerably more polluted surrounding. This study was carried out
during the summer season when heating plants/units, as a prevalent
source of air pollution in the city of Belgrade, are typically inactive.
Thus, traffic could be assumed as a dominant local source of pollution
including both exhaust and non-exhaust emissions. The results indi-
cated markedly lower air content of the traffic-related elements within
the garden in comparison to the street ambient (Fig. 3b, Fig. 1,
Supplementary material). The result implies that there were no relevant
local emissions (e.g., traffic) of these elements within the study area.
Some authors (e.g., Bukowiecki et al., 2009) pointed out by modelling
significantly higher concentrations of Sb and the other brake wear
related elements (Fe, Cu, Zn, Mo, Sn, and Ba) at the roadside location
than at the defined urban background sites. Loppi and Paoli (2015)
concluded that transplanted lichen Evernia prunastri reflected bulk
deposition of Al, As, Cu, Fe, Hg, Sb and Ti in the Botanical Garden of
the University of Siena (Italy), under conditions of relatively low air
pollution, and these elements originates from a traffic (Loppi and Paoli,
2015). Thus, the results of this study confirm the hypothesis that the air
pollution in botanical garden could be considered as an urban back-
ground, which influences the most of the citizens at any time. Known
background pollution level helps to recognize new pollutant sources
that are introduced into an area and the impact they may have on local
air quality. Estimation of the background level of air pollution in an
urban area could also be of great importance in air quality assessment
for better understanding the contribution of local sources to total
pollutant concentrations.
4. Conclusion
The moss (H. cupressiforme) bag biomonitoring of trace elements
confirmed the hypothesis that the Botanical garden could be considered
as an urban background area of air pollution. The results showed that
the moss element enrichment during the exposure periods was quite
low in spite of the downtown position of the study area.
Notwithstanding low trace element enrichment, the obtained results
clearly indicated that the moss exposed within the bags was under
stress, losing viability. The clear decrease of the pigment contents was
evident in the samples with the prolongation of exposure for the moss
that was exposed in situ in comparison to those kept in the phytotron.
Total phenolic content increased up to four times in the moss bags
exposed to ambient conditions in comparison with the moss kept under
the control conditions. The antioxidative capacity was the smallest in
the phytotron moss samples. Finally, the moss exposed in bags was in
stress, even under the homogenous-like conditions of the Botanical
garden, which certainly influenced active uptake of the elements and
favored mainly passive entrapment.
Acknowledgements
The authors are grateful to the Ministry of Education and Science of
the Republic of Serbia (Projects No. III43007, 173024 and 173030) for
financial support, and to the bilateral cooperation with Joint Institute
9
Urban Forestry & Urban Greening 25 (2017) 1–10
for Nuclear Research, Dubna, Russia.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.ufug.2017.04.016.
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