Introduction 

In the last decade, strategies and policies have been developed worldwide to improve air quality by addressing the factors that increase air pollution, aiming to protect the environment and human health. According to the World Health Organization ([49]), almost the entire global population (around 99%) breathes air with high concentrations of pollutants, and in some cases, the pollution exceeds WHO guideline levels. Ambient and household pollution is estimated to have caused 6.7 million premature deaths per year worldwide. Moreover, by the 2030s, the WHO has cautiously anticipated 250.000 additional annual deaths due to climate change and its consequences (⇒ https:/­/­www.­who.­int/­news-room/­fact-sheets/­detail/­climate-change-and-health). Due to pollution, 85% of annual deaths worldwide are premature deaths of people from low- and middle-income countries ([30]). However, people from high-income countries are also affected by pollution ([20]).

For this reason, many countries have rallied to reduce the effects of pollution. A turning point was the 1997 Kyoto Protocol, recognized as the most noteworthy environmental treaty ever negotiated for reducing greenhouse gas emissions ([8]). Actions like this are needed when voluntary agreements cease to have an effect. Such treaties provide some opportunities but mostly constrain the signatory countries ([43]). Strategies and policies have also been developed and implemented regionally. For example, the European Green Deal is an effort to reduce the net greenhouse gas emissions of the EU member states by more than half by the year 2030, compared to the levels reported for the year 1990 (⇒ https:/­/­commission.­europa.­eu/­strategy-and-policy/­priorities-2019-2024/­european-green-deal_en). Additionally, action plans like the “Towards Zero Pollution” for air, water, and soil plan are helping European Green Deal members achieve their targets (⇒ https:/­/­environment.­ec.­europa.­eu/­strategy/­zero-pollution-action-plan_en). According to the European Environment Agency’s publication “Air Quality in Europe 2022”, at least 238.000 people died prematurely in the EU that year due to exposure to pollutants (⇒ https:/­/­www.­eea.­europa.­eu/­publications/­air-quality-in-europe-2022). Common health impacts of pollution are stroke, ischemic heart disease, and primary cancer of the trachea, bronchus, and lung ([20]). In the spring of 2023, Romania enacted Law No. 97/2023 for the protection of remarkable trees ([34]). The law defines a remarkable tree as being at least 160 years old and having a minimum diameter measured at breast height (⇒ https:/­/­legislatie.­just.­ro/­Public/­DetaliiDocument/­267143). WIthin the EU, the Commission serves as a guardian to member states that do not comply with the relevant regulations and directives. For example, Germany, France, Belgium, Spain, Poland, and Romania have all recently been sent to the EU Court of Justice by the European Commission for failing to comply with relevant legislation on air quality. Several member states have also been fined ([18]).

According to a report from the Romanian National Environment Agency, published in 2022, Law No. 104/2011 is responsible for evaluating air quality in Romania and endorses/transposes all the directives of the European Parliament and the Council. The network for air-quality monitoring comprises 163 stations that monitor traffic and industrial emissions, urban and suburban pollution, and regional/rural pollution ([29]).

In urban environments, atmospheric pollutants with different sizes of particulate matter come from long-term anthropogenic sources. These sources include industrial emissions and fossil fuels combusted by vehicles and commercial and residential buildings. They affect the lives of urban residents and their green areas. Of these sources, fossil fuels used in vehicles are responsible for releasing most of the toxic trace elements, especially Pb ([17], [19], [40]).

Heavy metals, such as Cu, Fe, Pb, and Cd, are present in particulates of different sizes depending on the emission source. These particulates spread into the atmosphere of urban environments ([15], [3]). Even if cities banned cars from city centers, invested in pedestrian travel, cycling, and public transportation, or redesigned urban streets, it will take a significant amount of time before pollution levels decrease.

Recently, plants have been used as biomonitors or bioindicators to detect environmental pollution of the soil in both urban areas and forest ecosystems ([25], [51]). Trace elements are often present in leaves in higher quantities than in the soil ([26]). In particular, trees are good indicators of pollution due to their longevity, easy identification, and geographical distribution in urban areas ([1], [7]). Their longevity allows research/measurements to be repeated every few years over long periods. Heavy metals deposited on leaf surfaces can be absorbed and then spread into intercellular spaces, entering the channels, cuticles, and stomata. Moreover, toxic elements can be transported through plant xylem and phloem ([48]). The assimilation of heavy metals in trees is regulated by various factors, including the plant water deficit and leaf size, the light intensity, wind direction, and speed, and gas solubility in water.

Tree leaves are good indicators of heavy-metal pollution at a given time, reflecting what was recently absorbed from the atmosphere. Conversely, heavy metal pollution in the soil can take several years to build up and become detectable ([9]).

Many centuries-old, monumental trees are vital in the urban environment. They provide several benefits, such as carbon storage and sequestration and air pollution removal, and they help define the local identity of urban areas ([11]). Monumental trees often have more significant social and environmental benefits than smaller trees ([32]) due to the large number of leaves in their crowns. Indeed, monumental trees with a diameter of 76 cm can remove 30 to 65 times more air pollution in a year than smaller trees ([33]). Due to climate change effects, all European states have recently agreed to measure urban atmosphere heavy-metal pollution using plants as bioindicators ([40]). Various tree species have been used as bioindicators, such as Robinia pseudoacacia ([42]), Aesculus hippocastanum ([44], [45]), Eleagnus angustifolia, Fraxinus excelsior, and Juglans regia ([16]), Quercus ilex ([47]), Pinus brutia, Citrus aurantium, and Olea europaea ([39]), and Terminalia catalpa ([36]).

The main aim of this study was to provide a comprehensive overview of heavy-metal accumulation (Pb, Cd, Mn, Fe, Cu, Zn) in the leaves of (evergreen coniferous and deciduous) monumental trees from different cities located in nine counties in the Carpathian arch.

  Materials and methods 

Study sites

To analyze the effects of heavy metal pollution and its bioaccumulation in the leaves of monumental trees, twelve types of urban areas were selected across nine counties (Fig. 1).

Fig. 1 - Location of the monumental trees analyzed in this study (source: Google Earth™).

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The degree of exposure to pollutants in the studied urban areas is different due to the past and present factors. According to the Romanian National Institute of Statistics (⇒ https:/­/­insse.­ro/­cms/­en), almost 1 million inhabitants live in the study areas overall; the lowest number is in Jucu (5,349) while the highest is in Cluj Napoca (286,598). Also, a relevant criterion for the selection of trees to be sampled was their age and health status, two factors that can lead to variations in pollutant storage besides anthropogenic activities.

In total, leaf samples were collected from twelve sites: 2 from Alba Iulia county, 6 from Brasov county, 3 from Harghita county, 4 from Mures county, 2 from Sibiu county, 4 from Satu Mare county, 4 from Bistrita county, 3 from Maramures county and 9 from Cluj county (Tab. 1).

The majority of sampled trees were selected for their large size, while two specimens are from historical trees for that area (Fig. 2). The age varied between 100 and 600 years, the oldest trees being two Q. robur individuals from Blaj and Sibiu. The height ranges between 13 and 43 m, and the diameter between 0.68 and 2.13 m. The highest and the thickest tree was the monumental plane tree from Carei.

Fig. 2 - Examples of the monumental trees selected for the study.

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Sample collection and preparation

During the summer of 2022 (June-August), 10-20 g of fresh leaves (5-10 g of dry material) were collected from each of the 37 monumental trees selected. According to Cools & De Vos ([13]), mature leaves were sampled from the upper third of the crown. For the deciduous species, samples were collected during the second half of the growing season and before the onset of autumn yellowing and senescence.

The leaves were cut off the branches at a height of approximately 3 m using stainless steel scissors. Leaves were randomly selected from different areas of the crown, totaling about 30-40 fully developed leaves collected from each tree. The leaves were stored and transported in paper bags to the laboratory, where they remained until they were processed under laboratory conditions. In the laboratory, the leaf samples were left unrinsed and were oven-dried at 70 °C for 1 week. The dried samples were milled to uniform size using a laboratory mill, packed in polyethylene bags, and kept under stable conditions until their chemical analysis. The mill was washed after each grinding, first with alcohol and then with distilled water, to avoid any contamination between samples ([12]).

Chemical analysis

For the chemical analysis, 0.4 g (dry weight) of leaves was digested in 3 ml of 65% HNO₃ and 2 ml of 30% H₂O₂ in a microwave oven (SpeedwaveTM MWS-3, Berghof, Germany). After digestion, the solution was diluted with distilled water to a total volume of 25 ml. Cd (trace element) concentration was analyzed by inductively coupled plasma-mass spectrometry. Fe, Cu, Mn, Zn, and Pb concentrations were analyzed by inductively coupled plasma-optical-emission spectrometry ([38]).

  Results 

Lead

High lead (Pb) concentrations can negatively influence plant growth and development (e.g., root blackening), and can eventually cause plant death ([28]). The current acceptable Pb concentration for plants is 2 ppm ([35]), having previously been 3 ppm ([2]).

The lead concentrations in the monumental tree leaves ranged between 0 and 8.44 ppm (Fig. 3a). This range is below the toxicity threshold, but the acceptable threshold was exceeded in many trees. For example, in Miercurea Ciuc, the leaves of Ulmus glabra had a concentration of 2.8 times the threshold, with the leaves of Salix babylonica having a concentration of 1.8 times the threshold. In Blaj, the leaves of Tilia tomentosa had a concentration 1.7 times the threshold, whereas in Codlea city, the leaves of Acer platanoides had a concentration 2 times the threshold.

Fig. 3 - The metal content of monumental tree leaves. (a): Lead; (b): Iron.

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Iron

An iron (Fe) concentration of 40 to 300 ppm in tree leaves is normal, with less than 40 ppm being considered deficient and above 300 ppm being considered toxic, causing chlorosis (the abnormal reduction or loss of the green coloration in plant leaves - [6], [5]). However, the median Fe concentration in leaves is about 150 ppm ([5]).

High Fe levels were found in only two cities: Bistrita, in the leaves of Acer negundo (405.3 ppm, 1.3 times the toxicity threshold), and Cluj, in the leaves of Sophora japonica (320.7 ppm), Alnus glutinosa (430.3 ppm, 1.4 times the threshold), and Quercus robur (595.1 ppm, 1.9 times the threshold - Fig. 3b). These results indicate that Bistrita and Cluj had high levels of Fe pollution.

Manganese

Similar to lead Pb pollution, manganese (Mn) pollution is related to traffic density and vehicular emissions, although some researchers have indicated that airborne manganese Mn originates from soil ([37]). The Mn concentration in unpolluted natural environments should lie between 50 and 500 ppm ([5]).

The Mn concentration in the leaf samples ranged between 9.49 and 401.7 ppm, which is below the toxicity threshold (Fig. 4a).

Fig. 4 - The metal content of monumental tree leaves. (a): Manganese; (b): Copper.

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Copper

According to Bergmann ([5]) and Kabata-Pendias ([22]), tree leaf copper (Cu) concentrations of 5 to 20 ppm are normal, whereas less than 4 ppm is considered deficient. This can cause considerable changes to the biochemical processes in plants. Concentrations above 20 ppm are considered toxic for plant growth and, similarly to iron deficiency, can cause chlorosis ([6]).

The copper concentrations in the monumental tree leaves were low, ranging between 3.18 and 15.24 ppm (Fig. 4b). Only a single value exceeded the toxicity threshold (by 1.1 times) from an Alnus glutinosa in Cluj.

Zinc

The zinc (Zn) toxicity threshold in plants is 150 ppm. Levels above this threshold may cause production loss, while levels below 10 ppm may cause leaf deformation ([10], [31]).

The zinc concentrations in the monumental tree leaves were low in most locations (Fig. 5a), ranging from 5 to 111 ppm. This indicates that most of the locations were not polluted with this heavy metal. The highest zinc concentrations were found in Baia Mare, in the leaves of Camaecyparis lawsoniana (384.3 ppm) and Pinus strobus (234.3 ppm), and in Cluj, in the leaves of Populus nigra (192.7 ppm). These concentrations exceeded the toxicity threshold by 2.5 times, 1.5 times, and 1.2 times, respectively.

Fig. 5 - The metal content of monumental tree leaves. (a): Zinc; (b): Cadmium.

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Cadmium

The cadmium (Cd) concentrations in unpolluted natural environments should lie between 0.03 and 0.37 ppm ([2], [5]).

The cadmium concentrations in the monumental tree leaves were generally similar to the unpolluted levels, with most samples ranging between 0 and 0.22 ppm (Fig. 5b). Only three high values were recorded--in Baia Mare (2.92 and 2.78 ppm) and Cluj (0.79 ppm). These values exceeded the toxicity threshold by 8 times in Baia Mare and 2 times in Cluj.

  Discussion 

Lead

Lead has been used in different forms from ancient times. However, when it reaches high concentrations, the exposure to humans will impact almost all organs, especially the central nervous system, kidneys, and blood. Excessive levels will cause even death ([46]).

In the last decades, Lead pollution has been caused by anthropogenic activities and industrial emissions, but most is caused by emissions from road vehicles using fossil fuels ([37]). In general, lead appears on plant leaves by aerial deposition, especially along the roads, depending on traffic intensity and the distance from the road ([27]). The quantity of lead decreases in the leaves of plants with increasing distance from roads ([41]).

According to Kabata-Pendias & Piotrowska ([21]) in an urban environment, the normal concentrations of Pb in tree leaves are less than 10 ppm, this value is considered to be the toxicity threshold. According to WHO, a safety limit of lead for plants is 2 ppm ([35]) however in the past, an acceptable value of lead for plants can be also 3 ppm ([2]).

In Artvin, Turkey, no significant lead pollution was detected, as the Pb content in the leaves of Juglans regia ranges between 0.158-0.665 ppm ([16]) while in Serbia (Hall Pioneer Park, Belgrade), the highest Pb concentration found in plane leaves is of 13.748 ppm ([40]), exceeding of 1.3 times the toxicity threshold. Therefore, we concluded that there is no lead pollution in the cities where the samples were taken.

Iron

Iron plays an important role in respiration and photosynthesis reactions via enzymes and proteins ([4]). Iron derives from both anthropogenic and natural sources ([37]). In Belgrade, Serbia, high iron contents in pine leaves have been detected in the Hall Pioneer Park (319.411 ppm), whereas in Kosutnjak (also in Belgrade), the iron content was 185.411 ppm ([40]). In Artvin, Turkey, high iron concentrations have been reported in walnut leaf samples (332.5-698.2 ppm - [16]).

In the current study, the iron concentrations exceeded the limits only in the cities of Bistrita and Cluj, indicating Fe contamination in these areas.

Manganese

Manganese is a distinctive component of the photosynthetic oxygen-evolving system in chloroplasts ([12]), and manganese oxides have the role of fixing trace elements (cobalt, copper, zinc, nickel, and even pollutants like lead) in the soil. In medicine and some analyses, the chemical compound permanganate is used because it is a strong oxidizing agent ([2]).

Low levels of manganese, similar to those found in this study (53.6-349.2 ppm), have also been reported for various species, such as Robinia pseudoacacia from Denizli, Turkey ([12]), in the walnut leaf samples (1.001-204.6 ppm) from Artvin, Turkey ([16]) and in the Quercus ilex leaf samples (68.4-405 ppm) from Florence, Italy ([47]).

The data from all plots show values mostly below the 400 ppm threshold, indicating low pollution levels.

Copper

Copper is present naturally in the environment in the elemental form and is very important for the normal growth and metabolism of all living organisms, but it also can be commercially produced from sulfides and oxide minerals.

Copper is used in the manufacturing of electrical equipment, in construction, in agriculture (pesticides and fungicides), in wood preservation, etc. ([23], [24]).

In Edirne, Turkey, Cu content in the leaves of A. hippocastanum has been reported to range between 0.322-0.466 ppm ([50]), indicating a copper deficiency, while the leaves of Juglans regia in Artvin, Turkey, showed a Cu content ranging from 0.339 to 13.80 ppm ([16]). In contrast, in Athens, Greece, the average CU content in the leaves of Citrus aurantium, Olea europaea, and Pinus brutia was 99.86 ppm, which exceeds the toxicity threshold by 5 times ([40]).

In this study, all species except for one (Alnus glutinosa) sampled in Cluj city showed values below the maximum limit for copper, indicating low contamination levels.

Zinc

Zinc is an essential plant nutrient that plays an important role in the biosynthesis of enzymes, auxins, and proteins. The normal levels of zinc in plants are between 10 and 150 ppm ([2], [22]).

Low zinc concentrations have been reported in the leaves of Aesculus hippocastanum from Edirne, Turkey (0.374-0.532 ppm - [50]), and Ficus religiosa (0.766 ppm) and Eucalyptus species (9.290 ppm). All these levels are below the deficiency threshold and could induce leaf deformation. Conversely, optimal zinc concentrations have been found in Quercus ilex leaf samples (23.2-48 ppm) from Florence, Italy ([47]).

In the current study, zinc concentrations below the threshold were found in most locations. However, the samples from Baia Mare and Cluj, which have polluting industries, had concentrations exceeding the threshold by up to 2.5 times.

Cadmium

The toxic effect of cadmium on plants causes a reduction in plant growth and the appearance of chlorosis (abnormal reduction or loss of the normal green coloration of leaves). It is dangerous due to its great aerial dispersion, small size of particles being able to accumulate at great distances to the source of pollution ([14]),

In Athens, Greece, the mean cadmium content in tree leaves has been reported to be 0.62 ppm ([40]), exceeding the toxicity threshold 1.6 times. In the city of Edirne, Turkey, even in the roadside samples, the values of cadmium were very low (0.068 ppm - [50]).

Again, in this study, the highest values were found only in the cities of Baia Mare and Cluj, with values respectively 8 and 2 times greater than the toxicity threshold, indicating residual pollution in those areas.

  Conclusion 

The concentrations of heavy metals (Pb, Fe, Cu, Zn, Cd, Mn) were estimated under standardized experimental conditions in the leaves of 20 monumental trees of various species from urban areas in the Carpathian Arch. Overall, the results indicate low levels of atmospheric pollution. Only four of the six heavy metals analyzed exceeded the toxicity threshold, and only two of the nine counties included in the study had heavy metal pollution. The samples from Maramures County (in the city of Baia Mare) had zinc and cadmium levels above the toxicity threshold, and samples from Cluj County (in the city of Cluj) had iron, zinc, cadmium, and copper levels above the toxicity threshold.

Differences in heavy metal concentrations among tree species could result from their different capacities for heavy-metal storage. However, changes in leaves in response to concentrations of heavy metals in the atmosphere demonstrate that trees are suitable bioindicators of urban air pollution. Monumental trees can also aid in environmental restoration by decreasing pollution, resulting in healthier and more sustainable urban environments.

  Acknowledgments 

This work was supported by the National Institute of Research and Development in Forestry “Marin Dracea”, project no. 19070505 “Health status and conservation of monumental trees in Romania in the conditions of climate change”, within the BIOSERV program, financed by the Romanian Ministry of Research and Innovation.

  References