Ecological Indicators 126 (2021) 107357

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Ecological Indicators
journal homepage: www.elsevier.com/locate/ecolind

Country-scale complementary passive and active biomonitoring of airborne

trace elements for environmental risk assessment
Sorin Ștefănuț 1, Kinga Öllerer 1, *, Mihaela C. Ion, Larisa I. Florescu, Marian Constantin,
Cristian Banciu, Marilena Onete, Minodora Manu, Ioana Vicol, Mirela M. Moldoveanu,
Sanda Maican, Ioana Cobzaru, Roxana G. Nicoară, Elena D. Mogîldea, Dorina M. Purice,
Claudia D. Nicolae, Rodica D. Catană, Gabriela Teodosiu, Cristina A. Dumitrache,
Gabriel M. Maria, Ana-Maria Moroșanu, Ioana C. Paica, Constantin-Ciprian Bîrsan,
Gabriela Tamas, Mihnea Vladimirescu, Anca Manole
Institute of Biology Bucharest, Romanian Academy, 296 Splaiul Independenței, 060031 Bucharest, P.O. Box 56–53, Romania

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

Keywords: Despite the growing list of elements assessed and recognised as environmental and human health hazards,
Airborne metals pollution monitoring and mitigation strategies are mandatory only for a limited number of metals, for which
National survey ambient air quality limits are defined by national and international policies. In order to overcome this deficiency,
Tree leaves/needles
and promote cost-effective strategies and methods to assess airborne pollution, biomonitoring has been employed
Moss bags
Procedural deficiencies
on several occasions using various protocols and organisms. Here we present a complex national monitoring
Romania study based on a combined approach that integrates air pollution data obtained through passive biomonitoring
using the foliar content of various tree species and active biomonitoring with Hylocomium splendens moss bags.
Concentrations of seven air pollutant metals, Al, Cr, Co, Cu, Fe, Mn and Zn, not regularly monitored and lacking
established legal limit values, were measured in 410 passive and 142 active biomonitoring sampling sites across
Romania, in two seasons, representing the largest study reported up-to-date applying complementary passive and
active biomonitoring. Literature data reporting concentrations from uncontaminated areas was used to establish
the concentration limits for four pollution levels (no, low, medium and high pollution), in order to develop a
colour-coded warning system. High pollution levels were measured for 4.7% of passive and 9.8% of active
monitoring samples. The largest proportions of samples showing high pollution levels were obtained for zinc
(38.4%, for active monitoring) and chromium (18.9%, for passive monitoring). A significantly higher element
content was found in moss bag samples than in leaves/needles. Though we emphasize the potential of both
passive and active biomonitoring in assessing airborne pollution, our experiences and findings show that the
more standardized methodology of moss-bag biomonitoring is sounder and better suited for such large-scale
studies than a study conducted on multiple tree species. Some of the difficulties encountered during the two
biomonitoring studies are also reported, including the range limit of species and the lack of established reference
values for pollution limits.

* Corresponding author.
E-mail addresses: sorin.stefanut@ibiol.ro (S. Ștefănuț), kinga.ollerer@ibiol.ro (K. Öllerer), mihaela.ion@ibiol.ro (M.C. Ion), larisa.florescu@ibiol.ro (L.I. Florescu),
marian.constantin@ibiol.ro (M. Constantin), cristi.banciu@ibiol.ro (C. Banciu), marilena.onete@ibiol.ro (M. Onete), minodora.stanescu@ibiol.ro (M. Manu), ioana.
vicol@ibiol.ro (I. Vicol), mirela.moldoveanu@ibiol.ro (M.M. Moldoveanu), sanda.maican@ibiol.ro (S. Maican), ioana.cobzaru@ibiol.ro (I. Cobzaru), roxana.ion@
ibiol.ro (R.G. Nicoară), daniela.sincu@ibiol.ro (E.D. Mogîldea), dorina.purice@ibiol.ro (D.M. Purice), claudia.bita@ibiol.ro (C.D. Nicolae), rodica.blindu@ibiol.ro
(R.D. Catană), gabriela.teodosiu@ibiol.ro (G. Teodosiu), alina.coman@ibiol.ro (C.A. Dumitrache), gabriel.maria@ibiol.ro (G.M. Maria), anamaria.morosanu@
ibiol.ro (A.-M. Moroșanu), ioana.mihalache@ibiol.ro (I.C. Paica), ciprian.birsan@ibiol.ro (C.-C. Bîrsan), gabi.tamas@ibiol.ro (G. Tamas), mihnea.vladimirescu@
ibiol.ro (M. Vladimirescu), anca.manole@ibiol.ro (A. Manole).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.ecolind.2021.107357
Received 12 August 2020; Received in revised form 4 December 2020; Accepted 7 January 2021
Available online 25 January 2021
1470-160X/© 2021 The Author(s). Published by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).
S. Ștefănuț et al.
1. Introduction
According to the World Health Organisation, ambient air pollution
is provoking an estimated 4.2 million premature deaths per year
caused by stroke, heart disease, lung cancer and chronic respiratory
diseases, and 91% of the world’s population lives in areas where air
quality levels exceed WHO limits (WHO, 2018). In accordance with
this, ambient air pollution has been recognised for long time as the
main human and environmental health hazard, consequently a sys­
temic risk that has to be considered a priority issue, reflected in
practice by effective political, economic, environmental and social
decisions and measures (WHO, 2016; Tiwary and Williams, 2018).
However, despite the fact that in addition to WHO-monitored gaseous
pollutants (i.e. NO2, SO2, O3) and PM2.5, PM10, current airborne con­
centration levels of several elements are known to have a substantial
contribution to these health risks, the list of elements that are regularly
monitored and reported is limited to only a few (Directives 1999/30/
EC (European Council, 1999), 2004/107/EC (European Parliament
and Council, 2004) and 2008/50/EC (European Parliament and
Council, 2008; Tiwary and Williams, 2018; European Environment
Agency, 2019). As an outcome, we are generally lacking data on the
concentration dynamics of a significant number of pollutants repre­
senting human and environmental health risks. Furthermore, the ma­
jority of studies rely on concentration data measured only in a few
locations, partly because instrumental monitoring is conditioned by an
expensive infrastructure (Rivera et al., 2011; Vuković et al., 2015;
WHO, 2016; Capozzi et al., 2020).
On the other hand, biomonitoring is increasingly recognised as a
cost-effective alternative method that can contribute large-scale and
long-term quantitative and qualitative assessments, therefore a method
which could help overcome the financial and instrumental limitations of
direct monitoring (Wolterbeek, 2002; Markert et al., 2003; Harmens
et al., 2010; Ares et al., 2012; De Nicola et al., 2013; Vuković et al., 2015;
Urošević et al., 2017; Capozzi et al., 2020). The main drawbacks of
biomonitoring are the lower precision and the various procedural pit­
falls, including the less-standardized methodology, which is partly offset
by the typically much larger number of samples (Conti and Cecchetti,
2001; Capozzi et al., 2020). At the same time, its potential to provide a
more integrated, ecosystemic approach have been acknowledged also at
high decision making levels, in the case of tree leaves (European Com­
mission, 2013), while the European moss survey is used since 1990 in
the reports to the United Nations Economic Commission for Europe
(UNECE) Long-Range Transboundary Air Pollution (LRTAP) Convention
(Harmens et al., 2010).
Airborne trace element content of deciduous tree leaves/needles
proved to be a low-cost proxy of urban air pollution (e.g. Piczak et al.,
2003; Aničić et al., 2011; Sawidis et al., 2011; Tomašević et al., 2011),
but this biomonitoring method has been effective also at larger and more
diverse spatial scales (e.g. Aboal et al., 2004; De Nicola et al., 2013;
Capozzi et al., 2020). Despite the difficulties reported and the lack of a
generally accepted standardized methodology for foliar content mea­
surements, the concurrent conclusion of preceding studies (reviewed by
Tarricone et al., 2015; Gillooly et al., 2016) is that ‘passive’ bio­
monitoring using tree leaves/needles is an efficient method that corre­
sponds to the growing need for large-scale low-cost sensing of air
pollution.
One of the challenges in biomonitoring is represented by the diffi­
culty in addressing the diversity of environmental conditions, including
the bias caused by the range limit of species and their morphophysio­
logical differences, which often restrains the scale of the study (Tarri­
cone et al., 2015; Ștefănuț et al., 2018). This deficiency is overcome in
‘active’ biomonitoring, based on the translocation of the selected species
to the monitored area for a limited time, thus enabling the use of the
same species on a larger scale, independently of its natural occurrence,
at the same time granting a higher degree of standardization (Fernández
et al., 2000; Varela et al., 2016; Ștefănuț et al., 2019). A frequently
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Ecological Indicators 126 (2021) 107357
applied active biomonitoring method in terrestrial environments is the
one that uses moss bags (reviewed by Ares et al., 2014). Moss bag bio­
monitoring uses moss transplants collected from remote, unpolluted
areas, or cloned mosses, exposed for a predetermined period, and it is
based on the comparison of pollutant concentrations prior and after­
wards the exposure (Ares et al., 2012, 2014; Capozzi et al., 2017).
Regardless of the biomonitoring method applied, i.e. ‘active’ or
‘passive’ and the increasing number of such studies, the proportion of
those covering areas with multiple land uses remains relatively low (e.g.
Aboal et al., 2004; Makholm and Mladenoff, 2005; De Nicola et al.,
2013; Di Palma et al., 2017; Capozzi et al., 2020). The majority of
previous biomonitoring studies were conducted at a regional scale (see
ex. references cited above), and even those covering entire countries
were often based on a small number of sampling sites in relation to the
area covered (e.g. Harmens et al., 2010, 2015 – not all countries;
Capozzi et al., 2016). Here we report a biomonitoring study conducted
at a national scale, based on 410 passive and 142 active sampling sites
located in various land use types, encompassing road infrastructure,
urban/suburban, industrial, and rural areas, the latter including also
natural, conservation areas.
Considering the growing demand for air pollution monitoring as our
understanding of its health risks is developing, and the general tendency
of promoting ‘green’ technologies and methods, shown also by the
increasing number of biomonitoring studies, our main goal was to
conduct an exploratory biomonitoring study using tree leaves/needles
and moss bags. Following the initial biomonitoring studies addressing
the four compulsorily monitored and reported metals with established
limit values (Pb, Cd, Ni, As – Directives 1999/30/EC (European Council,
1999), 2004/107/EC (European Parliament and Council, 2004) and
2008/50/EC (European Parliament and Council, 2008), already pre­
sented in Ștefănuț et al., 2018, 2019, here we focus on seven metals
known for their potential health risks, but which are not all regularly
monitored in Europe, nor by the National Air Quality Monitoring
Network (NAQMN): aluminium (Al), chromium (Cr), cobalt (Co), cop­
per (Cu), iron (Fe), manganese (Mn) and zinc (Zn). These elements were
selected because previous surveys pointed out Romania as one of the
countries within Europe where the highest median concentrations for Al,
Cr, Fe and Zn can be found (Harmens et al., 2015), but for which there
are no limit values established regarding their human and environ­
mental health risks. The findings were integrated in the Long-term Na­
tional Monitoring System of Bioaccumulation of Airborne Heavy Metals
(BioMonRo), developed as an innovative warning and informing model
complementing the instrumental NAQMN (Ștefănuț et al., 2018).
Preceding studies have reported biomonitoring conducted simulta­
neously on tree leaves/needles and mosses sampled together from the
same sites (e.g. Aboal et al., 2004; De Nicola et al., 2013), and the
combination of passive and active biomonitoring was previously con­
ducted using the same species for both approaches in the case of aquatic
organisms (Lacroix et al., 2015; Ronci et al., 2016) or terrestrial plants
(Fernández et al., 2000). To our knowledge, excepting the study of
Capozzi et al., 2020, who used a combination of Robinia pseudoacacia
tree leaves and Hypnum cupressiforme moss bags for biomonitoring in
eight sites nearby Napoly, Italy, the study presented here is the first
country-scale assessment, and thus the largest study of this kind,
reporting airborne pollution levels based on complementary passive and
active monitoring, using tree leaves/needles and moss bags. Our aim
was to (i) obtain up-to-date concentration data for Al, Cr, Co, Cu, Fe, Mn
and Zn, metals known for their pollution potential, but not regularly
monitored, and (ii) to test the large-scale feasibility of passive and active
monitoring.
2. Material and methods
2.1. Sampling sites
In order to maximize data field collection and test effectiveness and
S. Ștefănuț et al.
suitability, we conducted both passive and active biomonitoring, using
tree leaves/needles in the first case and moss bags in the second.
For passive biomonitoring, the basis for designating the sampling
sites was represented by the 142 monitoring stations of the National Air
Quality Monitoring Network (NAQMN) distributed across the
41 counties of Romania. These monitoring stations were established
mainly in urban/suburban areas (45 stations) and industrial areas (57
stations), but also nearby traffic roads (31 stations), and in rural areas
(9 stations, including 3 control stations in isolated, natural areas). With
the aim to provide a better coverage, we have supplemented these
sampling stations with 268 additional passive biomonitoring sites,
resulting in a total of 410 sampling sites for passive biomonitoring,
meaning in average one station per each 580 km2. The aim was to have
at least 8 sampling sites in each of the 41 counties of Romania. These
additional sampling sites were partly in historically heavily polluted
industrial areas (101 sites), or in unpolluted, protected areas (47 sites),
while the rest were scattered across the country (Fig. 1). For active
biomonitoring we focused only on the 142 sampling sites corresponding
with the NAQMN stations.
2.2. Sampling species and sampling procedure
The experimental design and analysis were conducted according to
the protocol presented in Ștefănuț et al., 2018 for passive and Ștefănuț
et al., 2019 for active biomonitoring.
2.2.1. Passive biomonitoring
For passive biomonitoring, the purpose was to collect leaves,
respectively needles from generalist tree species regularly used in pre­
vious biomonitoring studies, such as those from the Populus and Pinus
genera, or Betula pendula and Aesculus hippocastanum (e.g. Djingova
et al., 1995; Aboal et al., 2004; Aničić et al., 2011; Sawidis et al., 2011;
Tomašević et al., 2011). However, Romania being the most bio­
geographically diverse member state of the European Union, as defined
by the European Environmental Agency, none of these species has a
country-wide distribution. As we intended to secure comparability of
data collected from different species and sampling sites as much as field
conditions made it possible, all sampled species were selected following
a literature survey, and have been used in similar biomonitoring studies
(Tarricone et al., 2015; Ștefănuț et al., 2018). Based on the literature
survey, we compiled a species list ordering the species according to
decreasing priority, and made decisions in the field on which one to
sample at each sampling site based on this list. Finally, we ended up
Fig. 1. The location of the 410 sampling sites. The 142 NAQMN monitoring
stations considered for both passive and active biomonitoring are marked with
grey dots, while the 268 sites considered for passive biomonitoring only are
marked black dots. Map created using ArcGIS.10.4. © 2016 Esri and
its licensors.
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Ecological Indicators 126 (2021) 107357
sampling from 32 tree species in total, to which, due to lack of trees in
certain sampling sites, we also added two herbaceous species, collecting
the above-ground parts of Phragmites australis (Cav.) Trin. ex Steud (60
samples) and Juncus effusus L. (3 samples). The most frequently sampled
tree species were Populus nigra L. (234 samples), Pinus nigra J.F. Arnold
(144), Betula pendula Roth (120), and Populus × canadensis Moench
(114). Further sampled tree species were Fraxinus americana L. (93
samples), Aesculus hippocastanum L. (84), Fraxinus excelsior L. (69), Picea
abies (L.) H. Karst. (60), Populus alba L. (33), Pinus sylvestris L. (33),
Populus tremula L. (27), Fraxinus angustifolia Vahl (24), F. pennsylvanica
Marshall (18), F. pallisae Wilmott ex Pallis (12), Acer negundo L. (12),
Populus sp. (12), Abies alba Mill. (9), Fagus sylvatica L. (9), Fraxinus ornus
L. (9), Pinus strobus L. (9), Populus × canescens (Aiton) Sm. (6), Populus
simonii Carrière (6), Platanus hispanica Münch. (6), Robinia pseudoacacia
L. (6), Acer campestre L. (3), A. platanoides L. (3), Morus alba L. (3), P.
pungens Engelm. (3), Quercus cerris L. (3) and Ulmus minor Mill. (3). One
of the reasons why we had difficulty in keeping the number of sampled
species lower, was that, for better accuracy, we decided to have three
sampling individuals in each sampling site, totalling 1230 samples
collected from the 410 sites.
Samples were collected in dry weather in July–October 2015, when
leaves/needles were matured and had sufficient time to retain the
airborne particles (Aboal et al., 2004; Tarricone et al., 2015) and con­
sisted of roughly 300 g leaves/needles, comparable in size and shape.
Only outer canopy leaves/needles were collected, from a height of 2 m
above the ground, and put into clean polyethylene bags, labelled and
barcoded. Sealed bags were transported at − 4˚C in cooler boxes, and
moved into a freezer, at − 20˚C.
As biomonitoring with tree leaves/needles can only be conducted
during the vegetation season, and considering the difficulties caused by
the range limitation of species, we decided to complement the passive
monitoring with the more standardized methodology of active bio­
monitoring. As this was intended as an exploratory study for testing the
feasibility of the biomonitoring methodologies, the two monitoring
studies were not simultaneous, but complementary, with passive
monitoring conducted in the vegetation season, and active monitoring
during late autumn–spring.
2.2.2. Active biomonitoring
For active biomonitoring, we selected Hylocomium splendens (Hedw.)
Schimp., a pleurocarpous moss species with well-defined annual growth,
frequently used in air pollution monitoring (Lucaciu et al., 2004; Blum,
2007; Rivera et al., 2011; Harmens et al., 2015; Ștefănuț et al., 2019).
The material for moss transplants was collected in 2015 from an un­
polluted, natural site, then cleaned in laboratory and left to dry at room
temperature.
Moss bags were prepared out of 2 × 2 mm polyethylene mesh cut in
12 × 12 cm pieces, which were each homogenously filled with 1 g dry,
devitalised apical green shoots of H. splendens, while a part of the ma­
terial was retained as control, to assess pre-exposure metal contents. At
each of the 142 NAQMN stations, five spherical bags were mounted at 2
m above the ground. Out of the five samples, two were selected
randomly for analysis per sampling site, totalizing a number of 284
samples, the rest being kept to allow the repetition of chemical analyses,
if needed, and to ensure the necessary number of samples in case of
damage by birds.
After initial heavy metal contents were measured, moss bags were
left outside for 4–7 months (between 126 and 224 days), from autumn
2015 to spring 2016, in order to cover the period with no vegetation, and
by this complementing the passive biomonitoring study (Ștefănuț et al.,
2018, previous section). These differences in the duration of exposure
were unintended and were caused by unexpected logistical difficulties
(i.e. impossibility to return at the appointed time to collect the samples).
Even with this, the exposure times were kept within the duration range
reported by earlier studies (reviewed by Ares et al., 2012), and corre­
sponded with the time length of passive biomonitoring. As exposure
S. Ștefănuț et al.
times were uneven for all bags, the control concentrations were
deducted from the obtained data and then concentrations reported for
one calendar year (365 days) were calculated for each sample, taking
into account the duration of the exposure. This was done on the basis of
the following formulae:
Conc_initial = the concentration in the control sample, not exposed
and measured prior to exposure;
Conc_determined = the concentration determined at the end of the
monitoring process;
Net_conc = Conc_determined − Conc_initial, the concentration
accumulated during the monitored period;
Annual_conc = Net_conc × 365/No. of days of sample exposure.
2.3. Preparation of samples and metal content analyses
2.3.1. Passive monitoring
An amount of 10 g of leaves/needles per sample was put in a clean
ziplock polyethylene bag, barcoded and sent for metal analyses to the
independent authorized I.M.U. Laboratories (www.imu-lab.com), where
samples were treated and analysed separately. Considering that our aim
was to detect element content, thus both bioaccumulation and adhered
depositions, the leaf samples were left unwashed (Sawidis et al., 2011;
Ștefănuț et al., 2018).
The analysis of the 1230 samples started with a preparation process
consisting of drying and granulation. Drying was realized through
heating to 50–60˚C, for 24 h. Dried samples were weighted in a per­
fluoroalkoxy jar. In a niche, 9 mL ultra-pure concentrated 65% nitric
acid and 3 mL ultra-pure highly concentrate 37% hydrochloric acid was
poured over the dry samples. The mixture was then left to rest at room
temperature, for the acid environment to oxidize the organic matter.
After one hour, the samples were moved into a digestion furnace
(MARSXpress SN/Mars 24050) and heated to 100˚C. Then, the samples
were removed into a niche and cooled down to room temperature, and
subsequently reinserted into the furnace and heated slowly, for five
minutes, until the mixture reached 175 ˚C. Following five minutes
digestion at constant heat, the jars were moved into the niche, where the
mixture was poured into volumetric flasks and left to rest till the solid
part settled down. For the quantification of metals, the supernatants
were moved into another volumetric flask, and diluted to 50 mL. The
samples were then analysed with an inductively coupled plasma mass
spectrometer (Agilent 7700 Series ICP-MS – ASX-500 autosampler),
which could detect element traces as low as 0.2 mg kg− 1. For quality
assurance, we used certified reference materials with documented
traceability: ICP multi-element standard solution IV – Certipur from
Merck, ICP/MS calibration standard No. 2, 3, 4, 5, and Ultragold Mer­
cury Standard from Ultra Scientific. Control samples were measured
before every batch. In order to determine the recovery values, the
samples were analysed for the seven targeted metals both before and
after adding the reference material. The acceptability criterion of values
was set in the 85–115% interval. The results for the average recovery
values on two levels were: Al – 103%, Cr – 94%, Co – 92%, Cu – 93.6%,
Fe – 108%, Mn – 91%, and Zn – 87%. For analysis and discussions, the
mean values per sampling site were considered. Concentrations were
reported as dry weight (mg kg− 1 dw).
2.3.2. Active biomonitoring
Metal content determination of the two moss bag replicates selected
randomly for each sampling site was conducted according to the same
procedure as presented above for passive monitoring. In a similar
manner as in the case of the leaf samples, the moss bag samples were also
left unwashed in order to avoid influencing the concentration of metals
(Aboal et al., 2011; Ștefănuț et al., 2019). In this case too, the mean
values per sampling site were considered.
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Ecological Indicators 126 (2021) 107357
2.4. Establishing pollution maximum values
2.4.1. Passive monitoring
In order to develop a colour-coded warning system, we used litera­
ture data to establish the concentration limits for four pollution levels
(no, low, medium and high pollution, Table 1). We selected studies
reporting concentrations from uncontaminated areas, measured in tree
leaves or needles and expressed in dry weight. We considered these
values as the upper limit of the now pollution level, and values
exceeding these limits as proofs of pollution. Then, in accordance with
the methodology for establishing the air lead pollution maximums
stipulated by the Decision No. 1600/2002/EC of the European Parlia­
ment and of the Council, the values exceeding the no pollution level
were split into three categories: exceeding the double of the natural
concentration limit value (high pollution degree; red), exceeding 70% of
the double and less or equalling the double of the natural concentration
limit value (medium pollution degree; orange), and exceeding the nat­
ural concentration limit value and less or equalling 70% of the double of
the natural concentration limit value (low pollution degree; yellow), as
shown in Table 1.
2.4.2. Active monitoring
In a similar manner as for passive monitoring, the concentration
values calculated according to the formulae presented in section 2.2.2.
were reported against the concentration limits established based on
reference values from literature. This was done by merging the eight
categories used in the pan-European study of Harmens et al., 2015 and,
for Co and Mn, not considered in that study, by referring to the values
reported through moss bag biomonitoring by previous studies (Vuković
et al., 2015; Urošević et al., 2017; Ștefănuț et al., 2019) (Table 2).
2.5. Visualising data
Concentration data were imported into a MS Access® database,
representing the basis of BioMonRo, a Visual Basic 6.0® software
developed by the Institute of Biology Bucharest (Ștefănuț et al., 2018) in
which pollution data obtained through passive and active biomonitoring
can be used either separately or simultaneously. The program processes
the concentrations and associates them simultaneously with the in­
tervals corresponding to the four signalling colours (green, yellow, or­
ange and red), and geographical coordinates of the sampling sites. For
both passive and active biomonitoring, it uses the mean concentration
values measured per sampling site. The BioMonRo software works on­
line, generating maps, graphs and reports that can be e-mailed auto­
matically to the responsible authorities (Ștefănuț et al., 2018, 2019).
2.6. Statistical analyses
We performed principal component analysis (PCA) to synthetize
our findings and see whether there are any correlations between the
studied elements. Prior of conducting PCA, we performed the Kaiser-
Meyer-Olkin (KMO) and the Bartlett’s sphericity tests to assess suit­
ability of data, i.e. whether the variables are correlated (Liu et al.,
2016). KMO values range from 0 to 1, and values > 0.5 are considered
suitable for PCA, while the Bartlett’s test of sphericity should have the
p significance<0.5 (p < 0.5) for a PCA (Liu et al., 2016). The Kaiser-
Meyer-Olkin test indicated that datasets were moderately suitable for
PCA for both types of biomonitoring, the KMO measure for sampling
adequacy value vas 0.59 for passive and 0.69 for active biomonitoring.
The small values of the significance level, p < 0.0001 for both moni­
toring types obtained for the Bartlett’s test statistic indicate that there
is a strong evidence against the null hypothesis of variables not being
correlated, thus the data are suitable for PCA (Liu et al., 2016). In order
to determine potential significant correlations among the studied ele­
ments, we calculated the Pearson correlation coefficients.
For the sampling sites used both in passive and active biomonitoring,
S. Ștefănuț et al. Ecological Indicators 126 (2021) 107357

Table 1
Passive biomonitoring – limits of Al, Cr, Co, Cu, Fe, Mn and Zn concentrations in the vascular plants, corresponding to the four
signalling colours used to produce the warning maps (Kabata-Pendias and Mukherjee, 2007; Kabata-Pendias, 2011; Sawidis et al.,
2011; Reeves, 2006).

Table 2
Active biomonitoring – limits of Al, Cr, Co, Cu, Fe, Mn and Zn concentrations in the moss tissues corresponding to the four sig­
nalling colours used to produce the warning maps (Harmens et al., 2015; Vuković et al., 2015; Urošević et al., 2017).

we performed one-way ANOVA followed by Tukey HSD post-hoc test to
check the significance of differences between concentrations measured
through the two monitoring techniques. The same analyses were con­
ducted to check the significance of differences between the concentra­
tions from sampling sites located in disparate land-use categories (rural,
urban/suburban, industrial, traffic – NAQMN categories).
For analysis, we used log (n + 1) transformed mean values per
sampling site. Analyses were conducted with the XLSTAT statistical
software, version 2014.2.06.
3. Results
The metal content of tree leaves/needles and moss bags showed
certain analogies. The proportion of samples marked with green,
included in the no pollution category was similar in the case of Al, Co, Fe
and Mn. Al, Cr, Fe and Mn exhibited larger proportions of samples

Fig. 2. The proportion of samples included in the four pollution levels for Al, Cr, Co, Cu, Fe, Mn and Zn measured through passive (left) and active bio­
monitoring (right).

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S. Ștefănuț et al. Ecological Indicators 126 (2021) 107357

signalling high pollution levels in passive than in active monitoring,
with a maximum recorded for Cr (18.90%). Cr was found in high per­
centages also for medium and low pollution. For all the other six metals
this value was below 5% (Fig. 2, left). High pollution was measured on
the whole in 4.7% of the tree leaf samples. This value was however much
higher in moss bags, accounting for 9.8% of the active monitoring
samples. In this case, the largest proportion of samples with concen­
trations exceeding the high pollution (red) maximum value was recor­
ded for Zn (38.03%), Cu (16.55%) and Co (11.97%). For all other metals
this value was below 1% (Fig. 2, right). The frequency of samples in
relation with the four pollution levels is presented as Supplement
(Fig. S1).
Moss bags accumulated larger amounts of the studied elements in
comparison with contents measured in leaves/needles, mean concen­
tration values measured per sampling site using active monitoring being
higher for all metals, except Mn (Table 3). The one-way ANOVA showed
that in sampling sites used in both passive and active biomonitoring
these differences were statistically significant for Co (F = 5.43, p = 0.02)
and Mn (F = 7.27, p < 0.0075). Highly significant differences (p <
0.0001) were found in the case of Al (F = 60.78), Cu (F = 30.90), Fe (F =
105.08,) and Zn (F = 61.47). For Cr differences were marginally sig­
nificant (F = 3.74, p < 0.054). Concentrations recorded in the leaves and
needles of four tree species commonly used in biomonitoring and most
frequently sampled in our study are shown in Table 4.
The one-way ANOVA showed that metal contents measured in
leaves/needles did not differ significantly between passive monitoring
sampling sites when considering land use categories. On the other hand,
accumulation levels recorded in active monitoring with moss bags
differed significantly between land use categories in the case of Cr (F =
3.86, p = 0.01), Cu (F = 16.81, p < 0.0001), Fe (F = 9.62, p < 0.0001)
and Zn (F = 4.16, p = 0.0075). The pairwise comparison conducted
using the Tukey HSD post-hoc test, with 95% confidence level, revealed
that these differences occurred mainly between traffic and urban/sub­
urban, and traffic and rural land uses, in the case of Cr, Cu, Fe and Zn,
while for Al, Co and Mn differences were not significant. For Cr, dif­
ferences were significant between traffic vs rural and traffic vs urban/
suburban land uses. For Cu, differences between traffic vs rural, traffic vs
urban/suburban and traffic vs industrial land uses proved to be highly
significant (p < 0.0001). For Fe, differences were highly significant for
traffic vs rural land uses, and significant for traffic vs urban/suburban,
traffic vs industrial, industrial vs rural, and urban/suburban vs rural
sites. In the case of Zn, differences were significant between traffic vs
urban/suburban land uses.
When visualizing the distribution of the sampling sites included in
the four pollution levels, there were several similarities between the
results for the two monitoring methods, revealing the most problematic
sites in terms of metal concentrations exceeding the maximums set for
high pollution levels (Fig. 3). The largest multiple for a maximum con­
centration recorded per sampling site to exceed the established pollution
maximum value was recorded for Zn, in active monitoring. With the
2490.79 mg kg− 1 maximum concentration it exceeded the 40 mg kg− 1
reference value set as lower maximum limit for Zn pollution by over 62
times, and the 120 mg kg− 1 lower maximum limit for high pollution by
over 20 times. There were only two cases in which the lower maximum
limit value for high pollution was not overrun in any of the sampling
sites, situation recorded for Al and Fe in active monitoring (Fig. 3).
The PCA analysis showed that in case of passive biomonitoring, the
strongest contribution to F1 was that of Cu (24.30%), Zn (20.19%), Cr
(19.48%) and Co (17.07%). Al (29.67%) and Fe (26.61%) had together
the strongest contribution to F2, while Mn (35.50%) accounted the
strongest contribution to F3, thus separated from the other elements.
This pattern was somewhat exhibited also by the active biomonitoring
data, where axes F1 and F2 accounted together for about 58% variance,
and Fe (24.76% contribution) and Al (21.05%) showed the strongest
association with F1, followed by Cu (18.84%), Cr (16.57%) and Zn
(12.11%). Mn was again separated, dominating in F2 (60.56%). The
PCA revealed thus a correlation between Cr and Cu, Co and Zn, and Al
and Fe (Fig. 4). The strength of these correlations was checked by per­
forming a Pearson correlation coefficient analysis.
For passive biomonitoring, the highest Pearson coefficients were
observed for Cu–Cr (r = 0.458), and Co–Zn (r = 0.365), and despite that
p values showed significance, the correlation coefficients were generally
lower in comparison with those for active monitoring (Table 5). On the
other hand, for active monitoring, the highest r values were related to
Fe: Fe–Al (r = 0.699), Fe–Cr (r = 0.600), Fe–Cu (r = 0.545) (Table 6).
4. Discussions
As far as we know, this biomonitoring study is among the largest ones
in terms of area covered (~238 000 km2) and number of sampling sites
(410, respectively 142), considering a wide range of land use types, from
remote reference sites to major pollution hotspots (Ștefănuț et al., 2018,
2019). Excepting Capozzi et al., 2020, who conducted both active and
passive biomonitoring in southern Italy, we are not aware of further
studies combining the two biomonitoring approaches using tree leaves/
needles and moss bags.
4.1. Element contents and monitoring method
Element concentrations and the proportion of samples marking high
pollution levels were higher in active monitoring for all metals (Table 3).
Moss bags accumulated larger amounts of the studied elements in
comparison with contents measured in leaves/needles, even with highly
significant differences in the case of Al, Cu, Fe and Zn. The finding is in
accordance with Aboal et al., 2004; De Nicola et al., 2013; Capozzi et al.,
2020, who concluded that mosses are more powerful bioaccumulators
than leaves and that the higher concentrations in mosses show that these
elements are mainly of atmospheric origin. However, it is important to
mention that in our case, contrarily to Capozzi et al., 2020, who also
used both active, moss bag and passive, foliar biomonitoring, it is not
possible to differentiate between the influence of the monitoring method
and seasonality as the two monitoring techniques were not simulta­
neous, active biomonitoring being conducted in autumn–winter, and
passive biomonitoring in spring-summer.

Table 3
Descriptive statistics for the mean concentration values of Al, Cr, Co, Cu, Fe, Mn and Zn (expressed as mg kg− 1) recorded per sampling site through passive (n = 410)
and active biomonitoring (n = 142).
1 1
Passive biomonitoring (mg kg− dw) Active biomonitoring (mg kg− dw)

Min. Max. Median Mean SD Min. Max. Median Mean SD

Al 0.58 486.88 29.72 41.74 45.11 0.00 3412.31 203.15 276.80 348.91
Cr 0.00 11.28 1.23 1.40 1.01 0.00 18.45 1.48 1.78 1.94
Co 0.00 9.96 0.20 0.51 0.88 0.00 15.90 0.12 0.91 2.06
Cu 0.74 21.12 5.67 6.16 2.84 0.49 95.88 8.16 12.18 12.66
Fe 11.81 910.01 130.10 149.36 89.18 0.00 3424.33 497.91 647.79 592.12
Mn 5.50 1168.01 55.62 95.28 128.82 0.00 447.96 80.11 88.16 58.28
Zn 5.61 1527.24 47.21 77.55 106.55 22.55 2490.79 96.02 230.74 365.15

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S. Ștefănuț et al. Ecological Indicators 126 (2021) 107357

Table 4
Minimum and maximum concentration values of Al, Cr, Co, Cu, Fe, Mn and Zn (expressed as mg kg− 1) recorded per sample with the leaves and needles of the four tree
species most frequently sampled in our study.
1
Species Value (mg kg− dw) Al Cr Co Cu Fe Mn Zn

Aesculus hippocastanum min 0.67 0.21 0.00 1.76 14.50 10.73 7.46
max 226.30 5.48 3.36 24.48 484.50 562.16 717.70
Betula pendula min 0.65 0.00 0.00 0.83 12.05 6.42 6.29
max 774.90 4.07 2.59 13.29 612.20 775.19 540.75
Pinus nigra min 1.20 0.19 0.00 0.44 7.93 1.89 0.00
max 256.12 8.96 2.19 16.73 503.24 232.12 2919.69
Populus nigra min 0.49 0.00 0.00 1.54 0.00 14.18 1.77
max 329.10 4.73 10.79 16.50 740.60 403.98 675.09

Fig. 3. The maps showing the mean concentration levels (mg kg− 1 dw) measured in 410 sites with passive biomonitoring using tree leaves/needles (left) and 142
sites with active biomonitoring using moss-bags (right) for Al (a, b), Cr (c, d), Co (e, f), Cu (g, h), Fe (i, j), Mn (k, l) and Zn (m, n).

Al, Cr, Fe and Mn had larger proportions of samples in the high
pollution category in passive monitoring, with a maximum recorded for
Cr (18.90%), Cr being found in high percentages in leaf samples also for
medium and low pollution (Fig. 2). This is consistent with the results of
Capozzi et al., 2020, who found higher deposition flux in Robinia
pseudacacia leaves than in Hypnum cupressiforme moss bag samples. The
differences might be caused by the fact that leaf contents sum up from
depositions and root uptake, but also by seasonal differences in emis­
sions (Harrison and Williams, 1982; Sun et al., 2014; Capozzi et al.,
2020). Meanwhile, Co, Cu and Zn exhibited significantly larger

7
S. Ștefănuț et al. Ecological Indicators 126 (2021) 107357

i j

k l

m n
Fig. 3. (continued).

proportions of samples in the high pollution category in active moni­
toring (Fig. 2), led by Zn (38.03%), in accordance with Vuković et al.,
2015, who found that the most enriched elements in moss bag samples
were these three traffic-derived pollutants. For Zn, all sites where high
pollution levels were measured in active biomonitoring were from
urban-suburban sites, except two, where traffic is heavier in winter.
Important amounts of anthropogenic Zn are related to vehicle emissions
and break and tyre wear, with higher values of total suspended
particulates and even 150% higher contents in winter than in summer
(Harrison and Williams, 1982; Sun et al., 2014; Vuković et al., 2015;
Capozzi et al., 2020).
Metal contents in tree leaves/needles vary greatly depending on the
sampled species, because of morphophysiological differences, in
particular leaf traits influencing particulate retention, but also on the
sampling period (Piczak et al., 2003; Aničić et al., 2011; Sawidis et al.,
2011; Tomaševic et al., 2011; Ștefănuț et al., 2018; reviewed by

8
S. Ștefănuț et al.
Fig. 4. Biplots of the first and second axis of PCA for the mean concentration values of Al, Cr, Co, Cu, Fe, Mn and Zn measured through passive (left, n = 410) and
active biomonitoring (right, n = 142).
Table 5
The Pearson correlation matrix for passive monitoring.
In grey – p values, in white – r values, bold indicates significant values.
Table 6
The Pearson correlation matrix for active monitoring.
In grey – p values, in white – r values, bold indicates significant values.
Tarricone et al., 2015; Gillooly et al., 2016). Therefore, as field condi­
tions did not enable the sampling of a lower number of species, the
comparison possibilities of metal contents between different sites are
very limited in the case of passive monitoring. Metal contents measured
in similar circumstances (species, month, land use category) can how­
ever be compared, therefore we sought to report our findings to previous
studies ensuring these conditions are met. From the literature surveyed,
we managed to find two studies that fulfilled all three criteria. Thus,
metal contents monitored with Aesculus hippocastanum leaves in urban
sites were overall consistent with those reported for the city of Belgrade
(Aničić et al., 2011; Tomašević et al., 2011). Thus, for Cr, Co, Cu, Fe and
Mn the range and mean values for metal contents found corresponded
with those reported by Aničić et al., 2011 and Tomašević et al., 2011. Al
9
Ecological Indicators 126 (2021) 107357
contents were however up to three times lower in Romanian urban sites
than those reported by Tomašević et al., 2011. For Zn, we found two
extreme values associated with well-known emission sources, the
smelters from the Baia Mare and Zlatna (Lucaciu et al., 2004). Excepting
these, the other values were in accordance with those reported by Aničić
et al., 2011 and Tomašević et al., 2011. For Betula pendula, metal con­
tents were in accordance with those found by Tomašević et al., 2011
excepting again Zn, for which values were overall twice as high in the
Romanian sites, but still below the 300 mg kg− 1 maximum set for high
pollution level.
In a similar manner as for passive biomonitoring, we also searched
for earlier active monitoring studies with moss bags to correlate our
findings. When comparing our results (minimum, maximum and mean
S. Ștefănuț et al.
values) for moss bag biomonitoring conducted in one of the main
pollution hotspots from Romania, the city of Baia Mare, with those of
Culicov et al., 2005 obtained by the same methodology, we found
significantly lower element contents for Al, Cr, Fe and Mn. In the case of
Co, the values were very similar, excepting the maximum values, which
were about twice as high in our case (0.69 vs 1.23 mg kg− 1). For Zn, both
minimum and maximum values were lower in our case, but means were
similar (122 ± 119 vs 22.49 mg kg− 1).
4.2. Evidencing pollution hotspots and correlations between pollutants
Interestingly, for the sampling sites considered in both monitoring
methodologies there was no overlap between the locations in which
maximum metal contents were measured, but only within some of the
sites included in the high pollution category (Fig. 3). This might be
caused both by the different sensitivity of the two methodologies, but
also by the differences in emissions between the summer and the winter
period (Harrison and Williams, 1982; Sun et al., 2014). However, there
were sites in which maximum concentration levels were measured for
more than one metal. One such location is Tașca cement factory, in
north-eastern Romania, where maximum values per passive monitoring
site for Al (1201.04 mg kg− 1), Cr (21.79 mg kg− 1) and Fe (1613.68 mg
kg− 1) were measured, with Populus alba leaves, explaining partly the
correlations found for these metals. Another such location is the city of
Oradea, where the highest mean values per site for Al (3412.31 mg kg− 1)
and Fe (3424.33 mg kg− 1) were measured in active monitoring. This
finding is unexpected, considering that the Al pollution source, the
Alumina refinery was shut down in 2002, and that element accumula­
tion measured through active biomonitoring is of atmospheric origin,
unlike passive biomonitoring assessing both depositions and uptake via
the root system. Besides being most probably a proof of pollution with
industrial dust from and abandoned industrial site, these results provide
a clear example explaining the strong correlations exhibited by the
Pearson coefficients (Table 4). Hunedoara, one of the largest and oldest
industrial centres from Romania, known for its highly polluting steel
mill (Lucaciu et al., 2004; Blum, 2007) proved to be the strongest source
for Cr (21.79 mg kg− 1) and Mn (248.65 mg kg− 1) in active monitoring.
This site was among the 10 strongest pollution sources also for Al, Fe and
Zn, thus our results are consistent with the previous reports (Lucaciu
et al., 2004; Blum, 2007) defining Hunedoara as an important pollution
hotspot.
The separation of Mn from the other elements in the PCA axes for
both active and passive biomonitoring might indicate different sources
or different environmental behaviour of Mn (Fig. 4). We presume that
the reason is most probably the different environmental behaviour, as
Mn was found in high concentrations together with other metals in
several samples, including those from Hunedoara, mentioned above.
4.3. Active biomonitoring data reflecting emissions inventory?
Out of the seven metals, only Cr, Cu and Zn are regularly monitored
within the European Union. According to the latest emissions inventory
report of the European Environment Agency, between 1990 and 2017,
Cr emissions dropped by 71% in the EU, in Romania with − 39%, Cu
emissions in the EU increased by 10%, in Romania with over 100%, and
Zn emissions in the EU dropped by 38%, and meanwhile increased by
9% in Romania (European Environment Agency, 2019). Such decreasing
trends have been shown also by the findings of the European moss
survey (Harmens et al., 2010, 2015).
In order to check whether our data fit these trends, we compared the
active, moss bag monitoring data, with those of the moss surveys re­
ported by Blum, 2007 for Romania and Harmens et al., 2015 for several
countries, including Romania, studies which assessed pollution levels
also by using Hylocomium splendens growing in natural conditions as a
biological material.
In case of Cr, there were fewer sites marking high pollution levels in
10
Ecological Indicators 126 (2021) 107357
comparison with Harmens et al., 2015, thus in accordance with the
decreasing trends shown in the inventory report (European Environment
Agency, 2019).
For Cu, no data was reported for Romania by Harmens et al., 2015,
while the comparisons with the findings of Blum, 2007 do not reflect the
reported trends.
Apart from Al, Cr and Fe, Zn was also mentioned by Harmens et al.,
2015 as an element for which Romania is one of the countries with the
highest median values. Despite using the same maximum values as
Harmens et al., 2015, there were significantly more sites marking high
pollution level is our case (57 vs 2). Thus the comparisons show an
increasing trend, however a stronger one than the 9% mentioned in the
inventory report (European Environment Agency, 2019). One explana­
tion for this sharper difference in our case might be the fact that the
mosses sampled by Harmens et al., 2015 were from unpolluted or
slightly polluted natural sites, while in our case the moss bags were
exposed mainly in urban/suburban areas and industrial areas.
5. Conclusions
The novelty of the presented study lies both in its scale, being the first
one applying complementary active and passive biomonitoring at a
national level, representing a complementary tool for the instrumental
monitoring network, and also partly in the applied methodology.
The fact that there was a lack of reference values, due to which we
had to establish pollution maximum values based on the procedure for
Pb and using several literature sources underscores the importance of
further experimentation with passive biomonitoring, and highlights one
of its main shortcomings, the lack of standardized procedures, which
would enable more reliable results, safeguarding its large-scale use.
Considering the difficulties encountered in conducting the passive
monitoring, in particular the high number of species which had to be
sampled due to the differences in field conditions occurring at such a
large scale, the overall results and the findings of the statistical analyses,
we conclude that active monitoring with a single moss species repre­
sented a more suitable assessment method in our case. Mosses have a
higher efficiency in accumulating atmospheric deposition, moss-bag
monitoring is a more standardized method, with the possibility to use
the same biological material over large areas and deduct initial metal
contents, thus more reliable for large-scale studies, where the diversity
of environmental factors ads further bias to data interpretation.
Furthermore, active biomonitoring offers the possibility to deduct initial
concentrations, providing more realistic data.
The advantage of passive biomonitoring is that it can be conducted
instantaneously, as it does not require the collection or laboratory work
with biological material before the sampling campaign (e.g. as shown by
the long-term and large-scale example of the European Moss Survey).
Besides, unlike moss-bag monitoring, reflecting only atmospheric
pollution, the foliar contents provide information both on accumulation
via roots and atmospheric depositions, consequently providing a more
systemic view of pollution. Furthermore, the method can be used on the
long term, and recorded concentration values can be considered as
reference value to monitor pollution trends in the future, with the
condition of using the same species and sampling period per site.
Both foliar accumulation and moss bag data provide a general
overview and up-to-date information on the pollution with metals which
are not regularly monitored in Romania, and the findings contribute to
the identification of areas representing potential human and environ­
mental health risks. Furthermore, the established sampling network, the
created database and the related BioMonRo system, already approved by
the Romanian Ministry of Environment, represent the basis of a cost-
effective tool for environmental assessment advancing mitigation
efforts.
S. Ștefănuț et al.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgements
The authors greatly acknowledge the help given by the technicians
both in the field and during the laboratory stages and the constructive
contribution of the two anonymous reviewers.
Funding
This study was supported through the project “Long-term National
Monitoring System of Bioaccumulation of Airborne Heavy Metals”
[grant number RO04-66074 – BioMonRo], financed through the EEA
Grants financial mechanism run by Iceland, Liechtenstein and Norway
under the Programme RO04 – “Reduction of hazardous substances”.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ecolind.2021.107357.
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