Atmospheric pollution is one of the most important problems in urban areas worldwide    having significant impacts on the population health, particularly in cities. In Europe, more than 85% of people are exposed to air pollution in exceedance of the European standards . Anthropogenic suspended particles in the atmosphere are usually associated with metals in airborne dust generated by traffic or industrial activity . Vehicular traffic is one of the most significant sources of heavy metal emissions in urban areas, which has increased in the last years   contributing by 57% - 75% to total emissions . Air particulate is rich in metals, such as chromium, cadmium, copper, iron, nickel, lead, and zinc that can be a health hazard when incorporated into the body through inhalation . Most of the toxic metals in the air are in form of fine particles, ranging from 0.003 µm to 10 µm .  identified the association of traffic-related air pollution and the increase in myocardial infarction events, considering that heart disease and stroke are the most common reasons for premature death attributable to air pollution for 80% of the cases . Nevertheless, vegetation that covers comparatively large segments of urban areas (i.e. private and public gardens, parks, sport fields, hedges and tree-lined avenues) is a valuable asset for modern cities delivering key functions and benefits . In particular, urban green spaces play an important role from a social perspective by promoting physical activity, increasing people interaction  and reducing stress factors. Moreover, plants may reduce metal pollution levels by accumulating particulate matter on their surfaces  . Particles can be deposited on plant surfaces and accumulated through sedimentation under the influence of gravity through impaction resulting from wind . A large amount of these particles are accumulated in internal leaf tissues  by absorbance via roots. The particles can be transferred from the leaf surface to the soil by rain . The topsoil has a large quantity of metals due to its high amount of organic substance . The extent of metals in urban soils depends also on the deposition time as well as the transport rate . As for plants, in comparison to annuals, trees trap and store more particles due to their larger total leaf surface area . Nevertheless, each species has a different capability of particles capture  evergreens being better traps for particles than deciduous species because of their longer leaf longevity, which can accumulate particles throughout the year . Moreover, deposition rates depend on air movement in the crown, transfer through the boundary layer adjacent to surfaces and the absorption capability of leaves. Thus, information on atmospheric pollution can be deduced from the concentration of specific substances in plant tissues offering low-cost information about the environmental quality  . Nevertheless, the basic criteria for the selection of plants for biomonitoring are that species should be present in a large number all over the monitoring area and widely spread, thus providing a high density of sampling points.
In such context, the main objective of this research was to analyze the metal accumulation capability by Platanusacerifolia (Aiton) Willd., Ailantusaltissima (Mill.) Swingle, Robiniapseudoacacia L. andQuercus ilex L., largely distributed in Rome. Rome is among the largest European cities with a large extension of the urbanized area (129.000 ha, of which 43,000 ha of greening and 50,000 ha of agricultural areas), 2,872,800 inhabitants , the dominant role of expansion and a large movement of public and private means of transportation (3,505,795; data from Automobile Club Italy 2017) which is a significant source of pollution emissions. The largest pollution emission occurs during the daytime peaking in the first hours of the morning when traffic density is the and from autumn to spring, decreasing in summer when traffic density decreases. Rome is also characterized by a high volume of green areas with important historical parks . The urbanization process in Rome has been increasing during the last years, and many new suburban areas have been built by scaling down free areas surrounding the city, which is changing into a mega city. For such characteristics, Rome may represent an ideal system to study the possibility of improving environmental quality by the selection of plant species that may contribute to lower pollution levels. Our data concerning P.acerifolia, R.pseudoacacia,A.altissima and Q.ilex metal accumulation capability can be incorporated into a geographic information system available for urban greening projects and can be exported to other urban areas.
2. Materials and Methods
2.1. Study Area and Climate
The study was carried out in Rome (41˚53'N 12˚29'E) in the period April-July 2019. Different types of sites were selected: historical parks (A sites) and high traffic density sites (B sites) (Figure 1 and Table 1). Traffic density (number of cars min−1) was monitored in the selected streets in June 2019 (mean value of the first 10 days of June) from 7:30 to 10:00 (peak hours, Gratani and Varone, 2013). In all the considered sites were present P.acerifolia (a naturalized species), R.pseudoacacia andA.altissima (two invasive alien species, IAS) and Q.ilex (a native species).The territory of Rome is between two distinct volcanic districts: the Albani Hills to southeast of the city and the Sabatini Mountains to northwest . The natural vegetation of the city consists of strips of persistent meadows of the suburban areas, trampled down environments, shrubs, ruderal or nitrophylous vegetation and fragments of deciduous and evergreen woods . Most avenues are characterized by Quercusilex,Quercus pubescens Willd., Platanusacerifolia and Pinuspinea L.
Rome is under a Mediterranean type of climate. The average total annual rainfall was 841 mm most of it distributed in autumn and winter. The average maximum air temperature of the hottest months (July and August) was 31.9˚C ± 0.4˚C and the average minimum air temperature of the coldest month (January) was 4.8˚C ± 1.0˚C. The mean yearly air temperature was 16.7˚C ± 6.6˚C (Figure 2). In the year 2019, from January to July, total rainfall was 430.5 mm with a peak in May (101.7 mm), the mean minimum air temperature was in January (3˚C) and the mean maximum air temperature in July (33.6˚C). The mean air humidity was 67.6% (Figure 3) (data provided by the Lazio Regional Agency for Development and Agricultural Innovation Meteorological Station of Rome, Lanciani Street, for the period 2008-2018 and for the study period in the year 2019).
Figure 1. Map of the considered sites in Rome. A sites: historical parks (A1 = Caffarella Valley Park, A2 = Borghese Historical Park, A3 = Doria Pamphjli Historical Park); B sites: high traffic level sites (B1 = Celio Hill, B2 = Oppio Hill, B3 = Passeggiata del Gianicolo Street). From Google My Maps.
Table 1. Description of the considered sites: A sites (historical parks) and B sites (high traffic density streets).
Figure 2. Climatic diagram according to Bagnouls and Gaussen (1953), for the period 2008-2018. The mean monthly air temperature (T˚) and the total monthly rainfall (R) are shown. The area between the two curves shows the period of summer drought. (Data provided by the Lazio Regional Agency for Development and Agricultural Innovation Meteorological Station of Rome, Lanciani Street).
Figure 3. Trend of the total monthly rainfall (R) and the mean air temperature (T) from January to July 2019 (Data provided by the Lazio Regional Agency for Development and Agricultural Innovation Meteorological Station of Rome, Lanciani Street).
2.2. The Studied Species
Platanusacerifolia (Aiton) Willd., Ailantusaltissima (Mill.) Swingle,Robinia pseudoacacia L. andQuercus ilex L. were studied.
Platanusacerifolia is a fertile hybrid, generated spontaneously among Platanusorientalis L., native to south-eastern Europe-Asia Minor and Platanusoccidentalis L., native to North America and introduced in Europe starting from the 16th century . P.acerifolia is particularly resistant to stress factors including pollution. It can reach 40 m in height and the crown has an expanded globular-oval shape. The root system is characterized by main roots deepening into the soil with numerous branches, and secondary roots expanding horizontally and more superficially . P.acerifolia was introduced in Rome in the late 1800s when the Piemontese Administration, in the ambit of the first regulatory plan of Roma Capitale (1873-1883), planted P.acerifolia trees in parks, squares and along avenues, particularly in those of the Lungotevere . Ailantusaltissima is a deciduous broad-leaved tree species native to North-Eastern China and North Vietnam . It is widely distributed throughout many temperate regions of the world including all continents with the exception of Antarctica .A.altissima was introduced in Europe in the late 17th century  and itis the most frequent non-native species in Italian cities . A.altissima is among the most tolerant species to high pollution levels .Itmay occur abundantly due to both an efficient sexual reproduction and a clonal growth. It is a shade intolerant species  although it can establish a ramet bank that can persist until a gap opens in the forest canopy; moreover, rapid establishment, growth and a vegetative reproduction in high light environments make disturbed areas such as timber harvests particularly prone to invasion by A.altissima . Allelopathic compounds in leaves, woods and roots  may exacerbate competitive exclusion of native species aiding in the formation of dense, monotypic stands. Robiniapseudoacacia is a deciduous broad-leaved species native to north America  which is considered one of the most invasive species worldwide , especially in Mediterranean countries . Originally introduced as a garden and landscape ornamental plant, this species has been widely planted in Europe  in the late 18th and early 19th century. The species was introduced in Italy in 1662 . Allelopathic substances produced in bark and roots  and flavonoids isolated from leaves (robinetin, myricetin and quercetin) can inhibit the growth of herbaceous species. It is a light-demanding species, which rapidly colonizes forest gaps from seedlings and by sprouting from stumps and roots, but it tends to be replaced after 15 - 30 years by larger and more competitive tree species for its limited competitive ability . R.pseudoacacia nitrogen-fixing capability facilitates the spread in urban areas where elevated CO2 concentrations lead to a marked increase in nitrogen fixation . Quercusilex is a broadleaved evergreen species native to the Central-Western Mediterranean Basin . Its natural distribution occurs from Portugal and Marocco to the Aegean Islands and Western Turkey, expanding northward up to Italy and France. Its altitudinal range is variable from the coast to 1800 m a.s.l. in southern Spain, and 2900 m a.s.l. in Marocco in the western part of the High Atlas . It is limited in its southern range by the longer summer drought  and in latitude by factors associated with low air temperature. Q.ilex occurring in mesic through xeric habitats is a shade-tolerant species  which is widely used for restoration of heavily deforested areas and abandoned croplands in the Mediterranean Basin. Q.ilex has been widely used as ornamental tree in parks, gardens and avenues in the city of Rome since the 16th century.
2.3. Tree Structure
Measurements of tree structural traits were conducted in June 2019. Trees were selected in the considered sites (three plants per each species and per each site). Tree diameter at breast height (DBH, cm) was measured by callipters (Silvanus callipter) and by a DBH tape when the diameter was greater than 65 cm. Tree height and crown height (H and h, respectively) were measured by electronic clinometers (Haglof, Sweden).
2.4. Leaf and Soil Sampling
Fully expanded sun leaves were collected at the beginning of July from the external portion of the tree crown (south-eastern side) for leaf morphological traits (10 leaves per each species and per each site) anatomical traits (10 leaves per each species and per each site) and for mineral content analysis (30 leaves per each species and per each site). Soil samples were collected near the selected trees (500 g per each site) after removing litter, at a depth of 0-15 cm, according to . Leaf and soil samples were stored in polyethylene bags and transferred immediately to the laboratory.
2.5. Leaf Morphology and Anatomy
The following morphological parameters were measured for each site: leaf area excluding the petiole (LA, cm2), obtained by the Image Analysis System (Delta-T Devices, UK), leaf dry mass (DM, mg), drying at 80˚C to constant mass. Leaf mass per unit of leaf area (LMA, mg∙cm−2) was calculated by the ratio between DM and LA. Total leaf thickness (LT, μm) was analyzed by a light microscopy using an image analysis system (Axiovision AC software).
2.6. Analytical Determination of Metals in Leaves and Soils
Leaf and soil samples were submitted to a pre-scanning treatment, according to . Leaf samples were oven dry at 70˚ for 4 hours, ground to pass 2 mm sieve. Compressed air was used to clean the grinder and sieve between new samples processing to prevent cross contamination. Soil samples were treated with the same procedure. All leaf and soil samples were placed in hermetic plastic bottles and transferred to the laboratory of the Department of Soil and Plant, Texas Tech University TTU (Lubbock, Texas, USA) for the scanning process by a Portable X-Ray Fluorescence (PXRF) Olympus Vanta M series PXRF on line power (110 VAC) featuring an Rh X-ray tube in a Geochemical mode. Scanning (dwell) time was 45 s per beam, 2 beams ere scanned sequentially such that one scan was complete in 90 s. Samples were massed on a 4 µm prolene thin film, placed on the sample stage in a test stand, covered with a Pb cup and scanned. The PXRF is a non-destructive proximal sensing instrument for the determination of elements in soil . The iron (Fe), copper (Cu), lead (Pb), zinc (Zn), strontium (Sr), cadmium (Cd), yttirium (Y), rubidium (Rb), thorium (Th), uranium (U), sulfur (S), arsenic (As), potassium (K), calcium (Ca) and manganese (Mn) concentrations were determined in leaf and soil samples from A and B sites. Detection limits for the elements are 5 ppm for Fe, Pb, Zn, Sr, Cd, Y, Rb, Th, As and Mn; 10 ppm for Cu; 25 ppm for K and Ca; 50 ppm for S (https://www.olympus-ims.com/it/vanta/#!cms[tab]=%2Fvanta%2Fresources).
2.7. The Biological Absorption Coefficient (BAC)
The Biological Absorption Coefficient (BAC) was calculated by the ratio between leaf and soil metal concentration) for all the considered species in the selected sites, according to .
2.8. Statistical Analysis
Data were analyzed by one-way analysis of variance (ANOVA) followed by the Tukey test for multiple comparisons to detect significance differences among each species collected in different sites. All statistical tests were performed using a statistical software package (Statistica 8.0, Statsoft, USA). The Pearson’s correlation coefficient was used to analyzed the relationship between the considered metals in leaves of the selected species.
3.1. Tree Structure
The structural traits of the considered trees species per each of the considered sites are shown in Table 2. The mean value of DBH, H and h for A and B sites were 1.76 ± 0.52 m, 17.55 ± 3.99 m, 12.93 ± 3.91 m, respectively, for A. altissima; 1.81 ± 0.41 m, 16.98 ± 2.98 m, 12.29 ± 2.55 m, respectively, for R. pseudoacacia; 1.91 ± 0.38 m, 18.73 ± 2.58 m, 15.74 ± 3.28 m, respectively, for P. acerifolia and 1.74 ± 0.06 m, 10.02 ± 0.80 m, 8.03 ± 0.94 m, respectively, for Q. ilex.
3.2. Leaf Morphology and Anatomy
The results of leaf morphological and anatomical analysis for all the considered species are shown in Table 3. LA was significantly higher for R.pseudoacacia, P.acerifolia,A.altissima in A sites (248.40 ± 77.13 cm2, 195.96 ± 25.58 cm2, 939.53 ± 98.21 cm2, respectively, mean value) than in B sites. Q.ilex had an opposite trend with the highest values in B sites (13.31 ± 3.10 cm2, mean value) than in A sites. DM had significant higher values in A sites (1.08 ± 0.39 g, 1.09 ± 0.27 g, 5.42 ± 1.22 g, mean value) than in B sites for R.pseudoacacia, P.acerifolia andA.altissima, respectively, Q. ilex having an opposite trend with the highest value in B sites (0.19 ± 0.06 g, mean value) than in A sites. LMA had significantly higher values for P. acerifolia in A sites (5.62 ± 1.38 mg∙cm−2, mean value) than in B sites, Q. ilex having an opposite trend with the highest values in B sites (14.08 ± 2.61 mg∙cm−2). A.altissima and R.pseudoacacia did not show significant differences in LMA between A and B sites. LT was significantly higher in A sites for R. pseudoacacia, A. altissima and P. acerifolia (135.13 ± 4.00 µm, 214.10 ± 5.24 µm, 215.30 ± 16.75 µm) than in B sites, Q.ilex having an opposite trend with a higher values in B sites (192.57 ± 41.20 µm) than in A sites.
3.3. Metal Concentration in Leaves
Leaf metal concentrations of the considered trees species in the selected sites are shown in Table 4. The leaf metals concentration related to traffic density was significantly higher in B sites for all the considered species. In particular, Q.ilex had a significant higher concentration of Fe, Cu, Pb, Zn, Sr and S in B sites decreasing by 36.5%, 51.5%, 40.0%, 29.9%, 54.3% and 43.0%, respectively, in A sites (mean value). P. acerifolia has significantly higher Fe, Pb, Cu and Cd values in B sites decreasing by 53.1%, 30.8%, 31.1% and 31.6%, respectively, in A sites, while Rb concentration was significantly lower in B sites increasing by 95.5% in A sites. R.pseudoacacia had significantly higher Fe, Cu, Sr and S values in B sites decreasing by 34.6%, 47.4%, 38.8% and 24.1%, respectively, in A sites (mean value).A.altissima had a significant higher Fe, Cu and Pb in B sites decreasing by 37.9%, 44.9% and 42.9% in A sites (mean value), while Mn was significantly lower in B sites increasing by 345.5% in A sites.
Table 2. Structural traits of the considered trees (mean value for each A and B sites): DBH = diameter at breast height (m); H = tree height (m); h = crown height (m) in the selected sites (mean value). A sites: historical parks (A1 = Caffarella Valley Park, A2 = Borghese Historical Park, A3 = Doria Pamphjli Historical Park), B sites: high traffic level sites (B1 = Celio Hill, B2 = Oppio Hill, B3 = Passeggiata del Gianicolo Street).
The Pearson’s coefficient analysis highlighted significant (p < 0.05) linear correlation among some of the considered metals (Table 5) in the selected species. In particular, in P. pseudoacacia Fe was positively related with Pb; Cu with S and Sr; Zn with Mn; Y with Mn; Th with Ca. In A. altissima Pb was positively related with Sr; Zn with S; Cd with U while Y was negatively related with Rb. In P. acerifolia Fe was positively related with Cu, Cd and S; Cu with Cd; Pb with Cd while Rb was negatively related with Fe, Cu, Sr and Cd; Th with U, and S with Ca. In Q.ilex, Fe was positively related with Pb; Cu with Sr, Th and S; Sr with S and Ca; Th with S; Rb with K while Cu was negatively related with Y; Pb with Rb; Y with Th and S.
3.4. Metals Concentration in Soil and BAC
The metal concentration in soil samples for the considered sites is shown in Table 6. Significant higher concentrations of Cu, Zn, Sr, Rb, Th, Ni, As, K and Ca were monitored in B sites decreasing by 54.8%, 33.9%, 43.3%, 24.7%, 29.7%, 34.4%, 30.4%, 12.9% and 47.3% in A sites. In particular, Celio hill had the highest soil metal concentration for Pb (828 ppm), Ni (31 ppm), Fe (46.119), Cu (255), S (1561) and As (49 ppm), and Celio hill and Oppio hill a higher concentration for Sr (1049 ppm and 1096 ppm, respectively).
The Biological Absorption Coefficient (BAC) was calculated for all the considered sites (Table 7) and it was significantly different among the species for metals related to traffic density (Fe, Cu, Pb, Zn, Sr and S) and for natural metals (Mn and Y). Moreover, among the species, Q.ilex had the highest BAC for Cu (0.244), Pb (0.014), Y (0.388) and U (3.347), followed by P.acerifolia for Fe (0.028), Th (0.784), S (5.973) and Mn (0.209), by A.altissima for Sr (0.126), Rb (0.328) and Ca (1.293) and by R.pseudoacacia for Zn (0.342) and K (3.526).
Contamination of aquatic and terrestrial ecosystems with metals is a major environmental problem. Although metals are naturally present in soils, contamination comes from local sources, mostly industry, agriculture, sewage sludge, waste incinerations and road traffic  . Heavy metals have been seen as a key marker since they are not degradable by natural processes . The contribution from different emission source sectors to air metals content depends not only on the amount of pollutants emitted, but also on the proximity to the emission source . Heavy metals accumulation in plant tissues causes various morphological, physiological and biochemical responses; however, these changes vary among species, which underscores that the response of plants to pollution is species-specific. Plants may be used as bioindicators of environmental pollution having the advantage of high temporal and spatial resolution  and leaves are the most sensitive plant organ to be affected by pollutants . The accumulation of contaminants in plants reflects the cumulative effects of environmental pollution from both soil and atmosphere . In particular, Zn is a natural element of soil but an excess of it is mainly due to vehicular traffic, losses of oil and cooling liquids, corrosion of galvanized steel safety fences . Fe and Cu are natural constituents of soil nevertheless, their source in street dust has been ascribed to corrosion of the metallic parts of cars like engine wear, thrust bearing and brush wear . Pb is a natural but minor component of the soil and is directly related to emissions from motor vehicles . For the last 40 years, road transport emissions were considered the principal source of Pb in urban areas being produced by combustion of petrol in the engine. In the eighties, Pb concentration in the atmosphere decreased because of the restriction in the use as an additive in gasoline in many Countries , and in 1989 in Italy. Cd is an additive in lubricating oils; Ni occurs both in organic and inorganic form in the soil , and in anthropogenic sources by its use in car brake discs and emission from cars with catalytic mufflers are the result of particles detached from the Al supporting substrate . The contribution to ambient S derives from sulphur-containing fossil fuels and biofuels used for domestic heating, stationary power generation and transport. Regarding As, Cd, Ni and Hg there was a decrease in their emission in Europe between the years 2000 and 2015 . Some effects on plants not directly caused by pollution but of which polluting agents are a predisposing factors can be observed, i.e. aphids grow more in polluted environments because pollutants increase the content of free amino acids  which represents a nutritional value for insects, consequently, plants are not able to activate mechanisms of defense .
Our results highlight differences in metals concentration in soil and leaf samples as well as in leaf morphological and anatomical traits for the considered species and among the considered sites. Leaf metals concentration in B sites (848, 26 ppm, mean values of all the metals for all the considered species) exceeds the concentration in A sites (601, 37 ppm mean value of all the metals for all the considered species). On average, the considered species show higher leaf concentration for Fe, Cu, Pb, Zn, Sr and S at Celio Hill and Oppio Hill (B sites) justified by the highest traffic density all day long. A lower leaf metal concentration at Passeggiata del Gianicolo Street, than the other considered B sites, can be justified by the larger open area of this B site, considering that pollutants are released at ground level and their upward movement is restricted due to tall buildings and congested thoroughfares, according to the results of  Among the considered species, Q.ilex and P.acerifolia have the highest concentration of metals determined by vehicular traffic than the other species. As regards to the soil, the highest concentration of Fe, Cu, Pb, Zn, Sr, Rb, S, Ni, Th, Ca and As were monitored at Celio Hill and Oppio Hill.The BAC, which reveals a variable and sometimes specificity of plants to absorb elements from the soil (51 Kabata-Pendias and Pendias, 2001) is the highest for metals related to vehicular traffic in Q.ilex forCu (0.244) and Pb (0.014); in P.acerifolia for Fe (0.028) and S (5.973); in A.altissima forSr (0.126), and in R.pseudoacacia for Zn (0.342) reflecting the different species ability to accumulate metals. The positive correlation among some metals confirms a common origin from vehicular traffic, according to . In particular, in P pseudoacacia Fe is related with Pb, Cu with S and Sr; in A. altissima Pb is related with Sr, and Zn with S; in P. acerifolia Fe is related with Cu, S and Cd, Cu is related with Cd, and Pb with Cd; in Q.ilex Fe is related with Pb, Cu with Sr and S, Sr with S. Moreover, leaves of the considered species which are exposed to traffic emissions show changes in some leaf morphological and anatomical traits, according to . In particular, P.acerifolia has significantly lower LA, DM, LMA and LT in B sites than in A sites, while Q. ilex shows an opposite trend, according to . A.altissima andR.pseudoacacia have significantly lower LA, DM and LT in B than in A sites. These results highlight the adaptive strategies of the considered species in response to environmental factors changing. In Q.ilex the increased LA, LT and LMA in response to pollution in B sites may be considered a compensatory response to the lower leaf longevity in these sites . Among the invasive alien species (IAS), A. altissima shows a lower leaf traits variability between A and B sites being one of the most tolerant species to high pollution levels .
The results as a whole underline the importance of tree species in urban areas for their ability to metals accumulation in leaves. Among the considered species, Q.ilex shows the highest metal accumulation capability. However, the large presence in Rome of invasive alien species (IAS), as A.altissima and R.pseudoacacia  Lucchese and Pignatti, 2009) negatively affects the presence of native species. In this case, the competent Authorities should intervene, in compliance with the Regulation EU n. 1143/2014 issued by the European Commission which included restrictions to the introduced IAS and an obligation to establish a framework of early detect and rapid removal of IAS, allowing the recolonization of the autochthonous species. In this case, Q.ilex is an autochthonous species that borders numerous avenues and historical parks in Rome contributing to improving urban air quality. Our data concerning tree structure, leaf morphological and anatomical traits and heavy metal accumulation capability could be used to realize an inventory available for urban tree planting programs to ameliorate air quality and to carry out a long-time monitoring of metal pollution for the city of Rome.
The authors are grateful to Professor D.C. Weindorf for performing the metals content analysis of leaves and soils by Vanta Olipmus M series PXRF analyzer.
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