Accumulation of heavy metals by wild edible mushrooms with respect to soil substrates in the Athens metropolitan area (Greece)

Accumulation of heavy metals by wild edible mushrooms with respect to soil substrates in the Athens metropolitan area (Greece)

Science of the Total Environment 685 (2019) 280–296 Contents lists available at ScienceDirect Science of the Total Environment journal homepage: www...

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Science of the Total Environment 685 (2019) 280–296

Contents lists available at ScienceDirect

Science of the Total Environment journal homepage:

Accumulation of heavy metals by wild edible mushrooms with respect to soil substrates in the Athens metropolitan area (Greece) Vasilis Kokkoris a,b,1, Ioannis Massas a, Elias Polemis b, Georgios Koutrotsios b, Georgios I. Zervakis b,⁎ a b

Agricultural University of Athens, Laboratory of Soil Science and Agricultural Chemistry, Iera Odos 75, 11855 Athens, Greece Agricultural University of Athens, Laboratory of General and Agricultural Microbiology, Iera Odos 75, 11855 Athens, Greece




• Metals content was assessed in six edible mushroom spp. growing in the Athens urban area. • High correlations were found among bioavailable (DTPA) metals content in soils and mushrooms. • K, Na and P concentrations in soils was associated to metals content in mushrooms. • The effect of soil texture on metals accumulation by mushrooms was speciesdependent. • Pb content in mushrooms exceeded the maximum tolerable daily intake.

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Article history: Received 10 April 2019 Received in revised form 28 May 2019 Accepted 29 May 2019 Available online 30 May 2019 Editor: Elena Paoletti Keywords: Available metal concentration Fungi Bioaccumulation Metal toxicity Soil properties Urban foraging

a b s t r a c t Six wild edible mushroom species, Agaricus bisporus, A. bitorquis, A. gennadii, Coprinus comatus, Psathyrella candolleana and Volvopluteus gloiocephalus, were collected from the Greater Athens area (Greece), together with their soil substrates (two depth-layers) for studying bioaccumulation of heavy metals in a densely populated urban environment. Total and bioavailable Cr, Cu, Fe, Mn, Ni, Pb and Zn concentrations in soils were assessed along with their respective concentrations in mushrooms, and were evaluated in conjunction with soil properties, including K, P and Na content, CaCO3 equivalent percentage, mechanical composition, pH and organic matter. In particular, Cu, Pb, Zn and Ni displayed a high variability in their total and bioavailable concentrations measured in the upper soil layer. Relatively high Pb and Ni contents were measured in mushrooms, while Cu, Zn, Fe and Mn concentrations varied considerably. No significant correlations were detected between total concentrations of heavy metals in soils and mushrooms, whereas bioavailable fractions for several metals were significantly correlated with their respective content in A. bisporus, C. comatus, P. candolleana and V. gloiocephalus. K, Na and P concentrations in soils were associated to the content of several metals in fruit-bodies. The effect of soil texture on metals accumulation by mushrooms was species-dependent since high correlations were found for V. gloiocephalus and C. comatus only. Interactions between metals content in fruit-bodies seem to be species-specific except for Ni vs. Fe and Mn vs. Fe, which are positively correlated in all cases. Overaccumulation of metals in fruit-bodies was established only in respect to the bioavailable fractions of Cu, Mn, Zn and Ni in soil. The levels of toxicity for Pb were exceeded in mushrooms of five species, whereas the rest of the heavy metals (with the exception of Cu) were detected at lower contents than their recommended dietary allowances or tolerable upper intake levels. © 2019 Elsevier B.V. All rights reserved.

⁎ Corresponding author. E-mail address: [email protected] (G.I. Zervakis). 1 Present address: Department of Biology, University of British Columbia, Okanagan campus, 3333 University Way, Kelowna, BC V1V 1V7, Canada. 0048-9697/© 2019 Elsevier B.V. All rights reserved.

V. Kokkoris et al. / Science of the Total Environment 685 (2019) 280–296

1. Introduction Soil functions as a filtering, buffering, storage and transformation system which receives and mediates pollution effects. While geological, geochemical and biological processes in the lithosphere require much time to develop, anthropogenic activities have a rather immediate impact on soils through atmospheric depositions and direct man-made interventions. Urban soils in particular suffer from the impact of traffic, waste disposal, heating and industrial emissions commonly resulting to the accumulation of various types of contaminants including high levels of heavy metals or metalloids also called potentially harmful elements (PHEs) (Ajmone-Marsan and Biasioli, 2010). Consequently, an urban environment is usually characterized by a mosaic of soils with marked variability in their properties due to natural spatial heterogeneity enhanced by human practices and are thus differentiated from naturally developed soils (Wei and Yang, 2009; Massas et al., 2010; Argyraki and Kelepertzis, 2014). On the other hand, the recent economic crisis which affected several countries worldwide in conjunction with modern trends in agriculture and food collection are gradually leading to the development of more resilient food systems based on the creation of city farm gardens and urban foraging (Barthel and Isendahl, 2013; Poe et al., 2014). Lately, there is a growing public interest in the harvest of wild edible mushrooms from urban and peri-urban environments since many of them are well-known for their gastronomic value, their high content in nutrients and health-promoting compounds, and their suitability for inclusion in low-calorie diets (Barros et al., 2008; Kalač, 2013). However, several studies have evidenced that mushrooms can accumulate high amounts of PHEs, especially when collected from heavily contaminated regions or soils with high metal content (e.g. mining sites, industrial areas etc.; Aloupi et al., 2012; Kojta et al., 2012; Árvay et al., 2017; Borovička et al., 2019); therefore, their consumption could have toxic effects on humans. Hence it is of importance to verify whether such foraging activities in and around urban areas are safe before being widely adopted. In general, accumulation of heavy metals by mushrooms is a complex process affected by both environmental (soil pH, content in metals, amount of organic material etc.) and intrinsic (taxon, developmental stage and mycelium age) factors; different species are behaving in a different way based on their nutritional requirements, but soil determines the availability and mobility of nutrients (Gadd, 2007). The rather few studies which examined the content of such pollutants in both mushrooms and their soil substrates seem to indicate that bioaccumulation varies considerably depending on the element and the fungal species (Giannaccini et al., 2012; Gucia et al., 2012; Falandysz et al., 2018). Still, pertinent information deriving from urban areas is scarce (Schlecht and Säumel, 2015; Svoboda and Kalač, 2003), while no data exist on the effect of underlying-soil characteristics (e.g. texture, pH, organic matter content, CaCO3 equivalent, content in K, P, Na and other elements) in respect to the accumulation of heavy metals by mushrooms. Equally important is that in most previous studies, total metal concentrations in soils were calculated, which are not always indicative of the metal potential availability to soil biota (Massas et al., 2010, 2013). Instead, the combined assessment of both total and available metal concentrations, and the interpretation of their relationships, could substantially confer at understanding metals effect on soil biological systems and at revealing the potential sources of recent/ongoing pollution events. For this purpose, wild mushrooms from six edible species, i.e. Agaricus bisporus (J.E. Lange) Imbach, A. bitorquis (Quél.) Sacc., A. gennadii (Chatin & Boud.) P.D. Orton, Coprinus comatus (O.F. Müll.) Pers., Psathyrella candolleana (Fr.) Maire and Volvopluteus gloiocephalus (DC.) Vizzini, Contu & Justo (Fungi, phylum Basidiomycota), were collected from several localities within the greater Athens area together with the respective soil substrates. Except of A. gennadii, all species are quite to very common in Greece (Zervakis et al., 1998; Konstantinidis,


2014). In particular, C. comatus, A. bisporus and A. bitorquis present a worldwide distribution and occur in subtropical and temperate regions; they are among the most common edible agarics in anthropogenic habitats, such as heavily disturbed soils, urban areas and livestock premises, while they are highly appreciated as concerns their edibility. P. candolleana is also a cosmopolitan mushroom consumed in many parts of the world (Boa, 2004); it is one of the most common species in gardens and parks, producing large numbers of basidiomes around wood litter, logs and stumps. V. gloiocephalus is also reported as widely distributed. However, its presence is so far confirmed in Europe and North America (Justo et al., 2011); it is popular as edible in Greece (particularly in the Aegean islands). Last, A. gennadii shows a relatively restricted Mediterranean distribution, its presence in Greece has been recently reported for the first time and it is considered as a rare species (Konstantinidis, 2014). The aim of this study was to determine the content of seven metals (Cu, Pb, Zn, Fe, Cr, Mn and Ni) in the fruit-bodies of six saprotrophic fungi, and to measure total and bioavailable metal concentrations in soil samples for assessing the type of relationships existing between metals bioaccumulation versus soil properties and content in various elements. Last, the issue of safely consuming such food from urban areas is examined vis-à-vis their possible toxicity to humans. Our main hypotheses were that wild mushrooms growing on urban soils would demonstrate higher concentration of heavy metals than their counterparts in natural habitats, and that elements content in fruit-bodies would depend on edaphic physicochemical characteristics and/or on their bioavailable fraction rather than on their total concentration in soils. 2. Methods 2.1. Study area The Athens Metropolitan Area (or Greater Athens including Piraeus) extends beyond its administrative municipal city limits, with a population of approx. four million people over an area of 412 sq. km. It spreads across a plain within the Attica basin which is surrounded by four mountains in the west, north and east (Mt. Egaleo, Mt. Parnitha, Mt. Penteli and Mt. Hymettus, respectively), and is only open to the south (Saronic Gulf). This large urban agglomeration was chosen for the present study since it hosts more than one third of the Greek population, is densely inhabited and shows high levels of air pollution due to traffic, residential heating, industrial and other emissions that are linked to many attributable deaths (Kalabokas et al., 2012; Fameli and Assimakopoulos, 2015; Mitsakou et al., 2019). In addition, when compared with other big cities, Athens presents increased amounts of the finer fractions of airborne particulate matter (PM) (i.e., PM10 and PM2.5; shown to be especially rich in Fe, Cu, Zn and Pb), which mostly derives from traffic-related air pollution (Valavanidis et al., 2006). Sampling was performed from 14 localities found mainly along a west-east transect crossing through the Athens Metropolitan Area; individual sampling sites were established within the aforementioned localities depending on mushroom occurrence, and they are all presented in Table 1 and depicted (per mushroom species) in Fig. 1: (1) the island of Salamina, on the west far-end of the Athens metropolitan area (#1 sampling site); (2) Perama, hosting several shipyards and small local industries (#1 sampling site); (3) Piraeus, the biggest harbor in Greece, with heavy ship and car traffic (#3 sampling sites); (4) Chaidari, on the west of the Athens metropolitan area, with several open areas and small woods but also adjacent to the avenue which serves as the main road exit to Peloponnese (#7 sampling sites); (5) Egaleo, a highly populated suburb with busy streets (#13 sampling sites); (6) Tavros, an off-center urban area with medium traffic (#1 sampling site); (7) Votanikos, on the edge of the city center area with rather heavy traffic (#12 sampling sites); (8) Kerameikos, near the historical city center with several open areas but with busy surrounding streets (#5 sampling sites);


V. Kokkoris et al. / Science of the Total Environment 685 (2019) 280–296

Table 1 List of all mushroom samples from the six species studied along with their corresponding code, collection locality/site and geographical coordinates, distance from the closest main (avenue) and secondary road, and type of locality from where they were collected. Species

Coprinus comatus (n = 9)b

Psathyrella candolleana (n = 14)

Volvopluteus gloiocephalus (n = 15)

Agaricus bisporus (n = 9)

Agaricus bitorquis (n = 6)

Agaricus gennadii (n = 5)

a b

Sample code CC1 CC2 CC3 CC4 CC5 CC6 CC7 CC8 CC9 PC1 PC2 PC3 PC4 PC5 PC6 PC7 PC8 PC9 PC10 PC11 PC12 PC13 PC14 VG1 VG2 VG3 VG4 VG5 VG6 VG7 VG8 VG9 VG10 VG11 VG12 VG13 VG14 VG15 AB1 AB2 AB3 AB4 AB5 AB6 AB7 AB8 AB9 AT1 AT2 AT3 AT4 AT5 AT6 AG1 AG2 AG3 AG4 AG5

Locality/site Tavros Sygrou-Fix Egaleo 1 Kerameikos 1 Chaidari 1 Chaidari 2 Chaidari 3 Kerameikos 2 Kerameikos 3 Egaleo 2 Egaleo 3 Egaleo 4 Egaleo 5 Egaleo 6 Kerameikos 4 Zappeion 1 Paleo Faliro 1 Piraeus 1 Egaleo 7 Votanikos 1 Kerameikos 5 Votanikos 2 Votanikos 3 Pedio Areos Piraeus 2 Perama Votanikos 4 Egaleo 8 Zappeion 2 Zappeion 3 Salamina Votanikos 5 Votanikos 6 Votanikos 7 Votanikos 8 Votanikos 9 Ilisia Votanikos 10 Zappeion 4 Zappeion 5 Paleo Faliro 2 Egaleo 9 Egaleo 10 Votanikos 11 Chaidari 4 Chaidari 5 Chaidari 6 Chaidari 7 Monastiraki 1 Monastiraki 2 Egaleo 11 Egaleo 12 Piraeus 3 Egaleo 11 Egaleo 12 Egaleo 13 Votanikos 11 Votanikos 12

Latitude 37°58′21.09″N 37°57′49.49″N 37°59′36.72″N 37°58′37.82″N 38°0′40.14″N 38°0′40.21″N 38°0′40.01″N 37°58′37.59″N 37°58′37.59″N 37°59′21.09″N 37°59′20.78″N 38°0′16.13″N 38°0′15.71″N 38°0′13.38″N 37°58′38.23″N 37°58′9.41″N 37°55′41.90″N 37°57′52.34″N 37°59′38.57″N 37°59′1.01″N 37°58′38.23″N 37°59′14.31″N 37°59′14.33″N 37°59′36.59″N 37°57′54.14″N 37°57′47.19″N 37°59′1.01″N 37°59′41.98″N 37°58′4.47″N 37°58′13.27″N 37°57′51.79″N 37°58′55.37″N 37°58′52.64″N 37°58′52.92″N 37°58′53.41″N 37°59′1.21″N 37°58′6.17″N 37°59′3.75″N 37°58′10.17″N 37°58′12.95″N 37°55′51.18″N 37°59′40.58″N 37°59′40.19″N 37°58′51.19″N 38° 0′9.14″N 38° 0′11.61″N 38° 0′15.41″N 38° 0′1.92″N 37°58′31.84″N 37°58′32.41″N 37°59′21.43″N 37°59′20.10″N 37°57′53.61″N 37°59′45.17″N 37°59′46.44″N 37°59′46.28″N 37°58′58.42″N 37°59′4.34″N

Longitude 23°40′46.42″E 23°43′34.60″E 23°40′36.60″E 23°42′54.11″E 23°38′39.20″E 23°38′38.94″E 23°38′40.01″E 23°42′51.60″E 23°42′50.88″E 23°40′46.42″E 23°40′46.19″E 23°40′32.05″E 23°40′31.44″E 23°40′30.84″E 23°43′14.17″E 23°44′11.22″E 23°41′9.68″E 23°36′48.99″E 23°40′34.57″E 23°42′18.16″E 23°43′14.17″E 23°41′44.53″E 23°41′44.55″E 23°44′14.84″E 23°36′50.65″E 23°33′41.92″E 23°42′18.16″E 23°40′36.76″E 23°44′3.99″E 23°44′11.67″E 23°26′44.35″E 23°42′12.41″E 23°42′24.70″E 23°42′22.76″E 23°42′21.46″E 23°42′18.24″E 23°46′55.42″E 23°42′12.33″E 23°43′54.66″E 23°43′56.82″E 23°41′10.65″E 23°40′38.33″E 23°40′38.61″E 23°42′25.80″E 23°40′27.45″E 23°40′29.48″E 23°40′31.80″E 23°40′27.42″E 23°43′12.01″E 23°43′11.22″E 23°40′46.59″E 23°40′45.88″E 23°36′48.36″E 23°40′40.66″E 23°40′40.72″E 23°40′44.72″E 23°42′20.30” 23°42′10.15″E

Distance to main road (m) Distance to closest road (m) Type of localitya 204 18 37 58 10 10 7 33 24 289 29 103 124 187 54 1 116 11 57 54 8 4 4 145 19 74 6 177 36 93 62* 203 126 129 132 12 191 7 1 3 66 145 147 155 330 250 125 514 222 226 84 115 34 200 235 145 17 2

26 18 37 12 10 10 7 33 24 13 18 22 14 2 54 1 116 11 57 54 8 4 4 145 19 11 6 76 36 44 62* 203 126 129 132 12 1 7 1 3 22 65 67 98 1 1 11 1 70 74 13 11 34 98 127 38 17 2


Type of locality, i.e. RV: road verge; PS/PG: public square and/or playground; US: unbuilt space; PV: pavement, P: park, CO: cultivated area – orchard. Number in parentheses below each species identity indicates the number of samples (mushrooms) examined.

(9) Monastiraki, a busy touristic site within the historical city center (#2 sampling sites); (10) Sygrou-Fix, at the city center with heavy traffic throughout the day (#1 sampling site); (11) Zappeion, a historical park and large green area within Athens city center (#5 sampling sites); (12) Pedion Areos, a large park in a highly urbanized area of Athens city center with heavy traffic (#1 sampling site); (13) Ilisia, on the eastern limits of the city center and in the vicinity of the suburban woods of Mt. Hymettos but influenced by the near-by highways (#1 sampling site); (14) Paleo Faliro, a densely populated suburb situated along the coastal avenue (#2 sampling sites).

2.2. Mushroom sampling and analysis For reasons of uniformity and for being able to compare among different localities for the same fungal species, sampling focused to the six most widespread edible mushroom species to be found at as many sites as possible (i.e., from 5 to 15) throughout the study area, in accordance to the outcome of preliminary mushroom occurrence/distribution surveys. Fruit-bodies (n = 58) produced by Agaricus bisporus, A. bitorquis, A. gennadii, Coprinus comatus, Psathyrella candolleana and Volvopluteus gloiocephalus were sampled from different types of urban habitats,

V. Kokkoris et al. / Science of the Total Environment 685 (2019) 280–296 Fig. 1. Sampling sites for each mushroom species examined, i.e. Agaricus bisporus, A. gennadii, A. bitorquis, Coprinus comatus, Psathyrella candolleana and Volvopluteus gloiocephalus. Individual sites are depicted by dots of different colours depending on the species sampled (as explained in the accompanying legend above) and presented in accordance to the localities/sites mentioned in Table 1.



V. Kokkoris et al. / Science of the Total Environment 685 (2019) 280–296

e.g., road verges, public squares and playgrounds, unbuilt spaces, and parks (Table 1). Mushroom species were identified as previously described (Zervakis et al., 1998; Polemis et al., 2012; Dimou et al., 2016). Fruit-bodies were collected by using a plastic knife and were superficially cleaned from soil and organic matter residues; they were then freeze-dried, pulverized and stored in a desiccator. One gram of the homogenized material was placed in a porcelain crucible and was ash-dried to 550 °C for 8 h. The remaining ash was dissolved with 5 ml nitric acid, suspended to 50 ml with distilled water, while a blank digest was prepared in the same way.

standard deviation, range of values, median and standard error. Although mean values are not to be used for compositional data (Reimann et al., 2008, 2012), they are hereby provided for facilitating comparisons with the outcome of previous pertinent studies. Especially as regards edaphic characteristics, descriptive statistics of the raw data include the mean, median, min, max, 10th percentile, 90th percentile and standard deviation values. The median is a robust estimator of central tendency while the lower and upper percentiles (in this case the 10th and 90th) are robust estimators of spread. In particular, the 10th and 90th percentile are commonly used to present edaphic data; they provide information about the value for which 10% or 90% of data points are lower than the 10th and 90th percentile, respectively.

2.3. Soil sampling and analytical procedures 3. Results and discussion From the sites where mushrooms were harvested, the respective soil substrates (n = 116) were also obtained as follows: for each fruit-body collected, three soil samples were taken from the upper- (0–15 cm) and from the lower-horizon (16–30 cm); the top organic layer including leaves, twigs and other debris was discarded. The three soil samples from each horizon were then pooled together, air-dried and sieved through a 2-mm sieve. The Bouyoucos hydrometer method (Bouyoucos, 1951) was used to assess the mechanical composition of soils, while organic matter (OM) content was determined by the Walkley–Black procedure (Nelson and Sommers, 1982). For pH measurements, 1:1 w/v soil to water ratio slurries were used and the CaCO3 equivalent percentage (eqCaCO3) was calculated from the evolved CO2 following HCl dissolution. For the determination of soil available P, the Olsen method was used as previously described (Olsen and Sommers, 1982), while the exchangeable forms of K and Na were determined by the ammonium acetate method (Page et al., 1982). Single step aqua regia digestion was applied to obtain the “pseudo-total” metal concentrations in soil samples (Gasparatos and Haidouti, 2001), while the DTPA (diethylene triamine pentaacetic acid) method was used for the determination of the bioavailable fraction of the studied metals (Lindsay and Norvell, 1978); in the text, these metal forms are termed “total” and “available” fractions, respectively. 2.4. Determination of metals in mushroom and soil samples – Estimation of bioconcentration factors Except of K, Na and P, all metal concentrations in mushrooms and soil extracts were determined by atomic absorption spectrophotometry by using a Varian - spectra A300 system. Exchangeable K and Na concentrations in all extracts were quantified using a PGI 2000 flame photometer (PG Instruments Ltd). Phosphorus concentrations in soil and mushroom samples were determined by a Shimadzu UV-1700 spectrophotometer. As reagent blanks, de-ionized water was used. To check accuracy and reproducibility, a control sample was analyzed for every 10 samples and 30% of the samples were reanalyzed. For AAS flame photometer and spectrophotometer determinations, the relative standard deviation of three measurements was lower than 3%. Bioconcentration factors (BCF) were calculated by dividing the concentrations of metals in mushrooms by their respective total and available concentrations in the underlying soil. 2.5. Statistical analysis Correlations and principal component analysis (PCA) were performed using R studio (Version 1.0.136 – © 2009–2016 RStudio, Inc.). Data was logarithmically transformed when needed to visualize the data in boxplots. Correlations were calculated after Spearman's coefficient which does not assume normality of the data. PCA was carried out after normalization of the data for 0.95 confidence level. For the edaphic and mushroom metal content, the descriptive statistics of the raw data were generated by using RStudio (Version 1.0.136 – © 2009–2016 RStudio, Inc.) and were presented in all cases as mean ±

Numerous studies were conducted in the past on the assessment of trace metal content in wild edible mushrooms (reviewed by Kalač, 2013). However, to our knowledge, this study is the first that examines explicitly wild edible mushrooms harvested within a major urban area and focuses in determining relationships between metal content of fruit-bodies to substrate properties such as soil physicochemical characteristics, total and available metals content, and habitat characteristics. 3.1. Soil properties and metal concentrations The sampling strategy led to results presenting a wide variation both in the soil characteristics and metal concentrations due to the heterogeneity of the investigated sites (i.e. distance from specific pollution sources, effects of various anthropogenic activities and land uses) but also because of human interventions which might have modified soil properties in roadside areas, parks and city squares. The physicochemical properties of the studied soils are presented in Table 2. According to median and percentile values, most soils showed medium to medium fine texture (L to SCL and CL) although some were classified as heavy (C) or medium light textured (SL). Soils were neutral to alkaline (pH: 6.95–8.30), and their eqCaCO3 was medium to high (33–476 g kg−1) apparently due to the nature of the parent materials prevailing in the area. The OM content varied greatly from very low to high values (0.55–17.63% d.w.); the extreme high values were obtained from cultivated tree orchards where organic amendments were most probably applied (sites within the campus of the Agricultural University of Athens). These characteristics are indicative of soils with varying cation exchange ability and adequate drainage, therefore with sufficient soil aeration and oxidizing conditions. Considering the typical Mediterranean rainfall distribution, such soil environments favor the proliferation of active biotic communities for most of the year (with the probable exception of the dry summer period for the top-soil part). In addition, the majority of the studied soils presented relatively high contents of available K and P (medians: 560 and 60 mg kg−1, respectively, for the 0–15 cm layer), while Na concentration was not particularly high (median: 257 mg kg−1) (Table 2). A clear trend of decreasing OM content and K, Na and P concentrations from the upper to the lower soil layer was observed (significant only for OM content and K concentration), whereas the opposite trend was detected for clay, eqCaCO3 and pH values (significant only for clay and pH). Considering the high OM content and K, Na and P concentrations in the upper soil layer and their substantial decrease in the lower soil layer in combination with the respective high 90th percentile values, such data are indicative of soils that in most cases have received some type of organic amendment and are enriched by the aforementioned nutrients. The descriptive details of the total concentrations for heavy metals are presented in Table 3. Among them, Cu, Pb, Zn, Ni and Cr displayed a notable variability in the values measured for the upper soil layer among different sampling sites (range: 462, 784, 724, 556 and 123 mg kg−1 respectively; maximum values being five to twenty-five

V. Kokkoris et al. / Science of the Total Environment 685 (2019) 280–296


Table 2 Values obtained after analysis of physicochemical properties in soil samples (n = 58) from two layers (upper: 0–15 cm; lower: 16–30 cm). Mean values sharing the same superscript letter are not significantly different (Gabriel's t-test, p b 0.05). Clay g kg−1

Silt g kg−1

Sand g kg−1

eqCaCO3 g kg−1

OM1 (%)

pH (1:1)

Exch.2 K cmolc kg−1

Exch. Na cmolc kg−1

P-Olsen mg kg−1

Upper soil layer Mean Median Min Max 10th perc. 90th perc. St. Dev.

225b 220 80 510 150 310 70

305a 305 220 400 230 370 47

470a 465 260 700 360 580 83

246a 237 41 431 129 381 92

7.33a 7.10 0.55 17.63 3.35 12.79 3.93

7.70b 7.74 6.95 8.30 7.38 7.98 0.27

568.3a 560.4 124.2 1283.2 183.6 1025.7 286.0

272.5a 257.2 47.2 947.2 76.0 495.1 190.0

73.71a 60.28 6.85 263.04 14.45 139.25 50.01

Lower soil layer Mean Median Min Max 10th perc. 90th perc. St. Dev.

262a 255 100 530 160 360 85

297a 310 120 450 200 370 60

441a 440 210 700 320 590 99

251a 242 33 476 135 361 93

4.93b 4.58 0.90 11.70 1.63 11.70 2.61

7.80a 7.78 7.31 8.24 7.44 8.15 0.25

436.1b 396.7 25.2 1160.0 104.4 788 286.3

231.9a 192.6 47.2 847.2 67.2 407.2 166.9

58.48a 42.35 0.80 262.42 16.56 97.04 49.76

1 2

OM: organic matter. Exch.: exchangeable.

times higher than respective minimum values). It is also of interest to note the large differences in the maximum concentrations detected for Cu, Zn and Cr between the upper and lower soil layers (i.e. 34%, 126% and 31% higher in the upper soil layer respectively) although their respective median values do not differ significantly. Given that the intervention values for Cu, Pb, Zn, Ni and Cr are 190, 530, 720, 210 and 380 mg kg−1 respectively (Netherlands M.H.P.P.E., 2000), the majority of sites presented relatively medium to low values for most of the metal concentrations measured. A notable exception was the Perama and Salamina localities, where high values were detected for almost all metals, i.e., 486, 320, 801, 120 and 105 mg kg−1 for Cu, Pb, Zn, Ni and Cr respectively for the upper-horizon (0–15 cm). Such results could be due to possible contamination of the particular area by atmospheric pollution since the respective values for the lower soil horizon (16–30 cm) were much inferior (79, 83, 160, 102 and 75 mg kg−1). Moreover, the sites in Chaidari and Egaleo presented relatively high values for Ni (often surpassing 100 mg kg−1; samples of the site ‘Egaleo 10’ exceeded 600 mg kg−1). However, only Zn total concentration was significantly higher in the upper soil layer, while no differences were observed for the other metals from samples of the upper and lower soil horizons. Noteworthy were also the high values detected in both soil horizons for Pb (713 to 801 mg kg−1) and Cu (187 to 215 mg kg−1) at two sites located within the campus of the Agricultural University of Athens (‘Votanikos 7’ and ‘Votanikos 8’) as well as in ‘Piraeus 2’ for Cu (264 and 362 mg kg−1 for the upper and lower soil layers respectively). In general, the 90th percentile of the surface soil samples was above the intervention limits for Cu, and in four sites (i.e., ‘Perama’, ‘Zappeion 2’, ‘Salamina’ and ‘Votanikos 7’ and ‘Votanikos 8’) the detected values exceeded them. On the other hand, maximum values for Pb and Zn are above the intervention limits only for ‘Votanikos 7’ and ‘Votanikos 8’, and ‘Perama’ respectively; the rest of the soil samples presented relatively low concentrations for these two metals when comparisons were based on median and 90th percentile values. Similarly, rather low values were observed for Ni and Cr, with the exception of the site ‘Egaleo 10’ as regards the former element. Previous studies assessing Cu, Pb, Zn, Ni, Mn and Cr total concentrations in top soils of the Athens area reported median values of 39 to 42 (this work: 47), 45 to 101 (99), 98 to 146 (174), 78 to 102 (90), 311 to 554 (456), and 84 to 141 (62) mg kg−1 respectively (Argyraki and Kelepertzis, 2014; Massas et al., 2010). When these concentrations were compared to those of surface soils from other urban areas around the world, similar values were detected for Cu, Pb, Mn and Zn, whereas higher values were found for Ni and Cr (as reviewed by Argyraki and

Kelepertzis, 2014) as it is commonly observed for Greek soils due to geogenic factors. As regards the concentrations of available metals, Cu, Pb, Ni and Zn presented distinctly large variability in measurements conducted in different sampling sites for the upper soil layer (range: 86.28, 61.75, 8.04 and 65.63 mg kg−1, respectively; maximum values being 20 to 80 times higher than minimum values measured) (Table 3; Supplementary material, Fig. S1). The 90th percentile values for many of the studied soils revealed relatively high available metal concentrations for Zn (30.55 mg kg−1), Pb (14.52 mg kg−1) and Cu (26.11 mg kg−1), which are indicative of soil enrichment and their potential accumulation in fungal and plant tissues. Such results could be attributed to rather recent soil enrichment by metals not yet allowed to become sequestered and strongly absorbed by the soil colloids. The latter hypothesis is also supported by the depth distribution of mean available metal concentrations, i.e. they were higher in the upper soil layer and demonstrated significant differences (between the upper and the lower soil layers) for Zn, Mn, Fe and Ni. According to median concentration values and in terms of soil fertility, the availability of Cu, Zn and Mn was adequate (in most cases it was b10% of the respective total content), while the availability of Fe was very low (b1‰), presumably due to the relatively high eqCaCO3 content in the soils examined. Although in the majority of pertinent studies, total metal concentrations were used for soil pollution assessment and as indicators of longterm soil enrichment (Facchinelli et al., 2001; Wang et al., 2005; YaylaliAbanuz, 2011), such measurements provide negligible information on metals availability. In contrast, the determination of the available fraction of metals in soil could provide valuable data related to recent metal depositions and on the metals potential to sequester and/or get absorbed by the soil biota (Massas et al., 2009, 2010). Concentrations of available metals in the studied soils demonstrated similar tendencies to what was observed for total metal measurements in individual sites. Hence, relatively medium to low values were noted in the majority of the localities examined with the exception of ‘Perama’, where high values were measured for Cu, Pb and Zn in the upper soil layer (i.e. 64.10, 61.75, 65.63 mg kg−1) in contrast to the considerably inferior values detected in the lower soil layer (10.54, 8.78 and 10.28 mg kg−1 for Cu, Pb and Zn, respectively). The ‘Salamina’ site demonstrated Cu concentrations for both the upper and lower soil layers (56.63 and 86.28 mg kg−1, respectively). Moreover, the ‘Chaidari’ and ‘Egaleo’ sites presented the highest values for Ni (in most cases exceeding 1.6 mg kg−1 and reaching up to 8.04 mg kg−1). For both soil layers, total and available Cu, Zn, Pb and Ni concentrations were positively correlated (p b 0.05) pointing to common origin


V. Kokkoris et al. / Science of the Total Environment 685 (2019) 280–296

Table 3 Metals content in mushrooms, and total and available metals concentration in underlying soil. The values provided correspond to mean ± SE and bioconcentration values (in brackets), range (in parentheses) and median value. Zero values represent concentrations below detection limit. Metal


(mg kg−1 dw) Pb


0–15 cm soil total metal 16–30 cm soil total 0–15 cm soil available metal 16–30 cm soil available Cu


0–15 cm soil total 16–30 cm soil total 0–15 cm soil available 16–30 cm soil available Mn


0–15 cm soil total 16–30 cm soil total 0–15 cm soil available 16–30 cm soil available Zn


0–15 cm soil total 16–30 cm soil total 0–15 cm soil available 16–30 cm soil available Fe


0–15 cm soil total

Species (no. of samples examined) Coprinus comatus (n = 9)

Psathyrella candolleana (n = 14)

Volvopluteus gloiocephalus (n = 15)

Agaricus bisporus (n = 9)

Agaricus bitorquis (n = 6)

Agaricus gennadii (n = 5)

5.64 ± 1.28 (0–11.35) 6.70 76.50 ± 8.31 [0.07] (37.48–117.6) 76.08 73.25 ± 6.25 (36.40–104.68) 72.68 7.90 ± 1.37 [0.71] (2.74–12.43) 7.43 8.55 ± 1.41 (2.12–13.25) 7.09

5.17 ± 1.39 (0–16.81) 7.46 90.39 ± 11.39 [0.06] (46.93–164.60) 73.16 93.54 ± 14.71 (42.85–193.75) 66.6 7.52 ± 0.97 [0.69] (2.98–13.68) 6.68 8.68 ± 1.37 (2.91–18.48) 6.76

8.43 ± 1.35 (0–16.31) 9.00 209.76 ± 59.30 [0.04] (49.78–783.75) 132.1 191.98 ± 64.26 (35.73–800.63) 104.95 14.34 ± 3.67 [0.59] (3.45–61.75) 11.03 10.93 ± 1.85 (2.08–23.58) 8.78

4.23 ± 1.26 (0.5–11.21) 3.61 148.61 ± 42.47 [0.03] (25.38–350.33) 102.45 159.58 ± 41.47 (29.63–391.22) 106.75 4.53 ± 1.18 [0.93] (0–11.23) 5.49 5.88 ± 1.15 (1.01–10.75) 5.76

2.80 ± 0.64 (0.74–4.86) 2.31 73.42 ± 18.36 [0.04] (25.60–128.72) 69.74 197.16 ± 71.23 (37.98–418.72) 119.39 4.26 ± 1.71 [0.66] (1.01–12.46) 2.63 7.45 ± 2.21 (1.47–12.75) 7.96

4.12 ± 0.57 (2.55–5.34) 4.88 145.32 ± 39.33 [0.03] (56.64–292.57) 123.95 127.81 ± 33.92 (40.61–223.10) 156.92 7.92 ± 3.21 [0.52] (2.45–20.23) 6.69 7.17 ± 2.83 (2.16–17.82) 6.30

56.82 ± 6.03 (31.75–90.56) 52.30 31.70 ± 2.66 [1.79] (23.25–50.25) 32.88 32.65 ± 2.96 (22.85–50.43) 32.86 3.17 ± 0.53 [17.92] (1.07–5.30) 3.08 2.62 ± 0.53 (0.73–4.84) 2.91

45.88 ± 4.43 (16.08–90.35) 47.39 43.62 ± 3.73 [1.05] (29.15–68.85) 38.16 39.77 ± 4.32 (27.58–75.95) 33.05 3.70 ± 0.49 [12.40] (1.47–7.10) 2.95 4.03 ± 0.82 (1.21–11.02) 2.79

39.54 ± 4.26 (22.75–70.12) 35.20 131.24 ± 38.55 [0.30] (24.30–485.5) 46.35 92.53 ± 25.21 (21.10–362) 44.35 16.36 ± 5.36 [2.42] (0.80–64.10) 5.64 14.35 ± 5.82 (0.70–86.28) 4.15

65.81 ± 13.22 (37.20–165.66) 51.34 67.38 ± 9.52 [0.98] (31.9–109.87) 69.86 69.51 ± 14.81 (30.75–160.43) 63.78 1.69 ± 0.25 [38.94] (0.74–2.87) 1.83 1.81 ± 0.45 (0.37–4.4) 1.20

64.81 ± 12.11 (38.98–113.08) 51.19 66.00 ± 4.37 [0.98] (50.23–76.13) 68.58 124.75 ± 42.47 (32.35–257.91) 68.84 1.57 ± 0.43 [41.28] (0.87–3.63) 1.25 3.36 ± 1.21 (0.75–6.88) 2.37

21.75 ± 9.05 (5.47–49.72) 8.33 92.50 ± 9.62 [0.24] (64.97–117.55) 98.75 89.27 ± 3.75 (77.82–101.48) 88.66 2.09 ± 0.30 [10.41] (1.23–3.02) 2.15 1.69 ± 0.17 (1.15–2.03) 1.86

11.30 ± 2.37 (3.75–24.48) 7.50

13.67 ± 2.07 (6.65–33.04) 10.28 464.35 ± 23.66 [0.03] (377.75–691) 458 486.32 ± 23.65 (380.50–666.75) 460.25 11.81 ± 2.10 [1.16] (1.27–24.03) 7.38 9.48 ± 1.38 (1.04–16.20) 7.58 92.76 ± 7.96 (45.92–155.27) 86.98 226.81 ± 24.35 [0.41] (84.75–323.25) 256.50 179.57 ± 22.27 (83.50–325.63) 159 15.98 ± 3.39 [5.81] (2.26–38.73) 11.99 12.34 ± 2.82 (0.89–30.08) 6.68 344.95 ± 51.72 (117.55–697.97) 323.93 20,971.43 ± 1729.78 [0.02] (14000–36,237.50)

27.29 ± 2.74 (12.80–49.92) 25.28

35.88 ± 2.89 (28.81–54.72) 32.41

43.93 ± 5.47 (32.74–59.14) 38.72

488.34 ± 33.91 [0.06]

465.95 ± 27.85 [0.08]

(319.25–662.50) 436.50 495.96 ± 33.91 (320.75–732.50) 436.50 8.49 ± 1.29 [3.21] (2.14–18.13) 7.61 6.49 ± 0.83 (1.18–15.03) 5.67 77.93 ± 2.44 (55.75–95.60) 79.48

(349.32–610.37) 500.63 478.45 ± 34.97 (366.42–679.38) 453.69 2.38 ± 0.82 [15.08] (0.89–6.79) 1.27 1.98 ± 0.83 (0.93–8.63) 1.15 80.54 ± 11.11 (40.93–142.04) 71.05

187.25 ± 24.23 [0.42]

179.40 ± 12.92 [0.45]

(77–800.52) 154.63 166.94 ± 19.98 (79.75–313.80) 159.5 16.83 ± 4.18 [4.63] (3.90–65.63) 11.1 11.87 ± 3.6 (2.33–36.10) 5.42 385.28 ± 79.23 (81.85–1024.63) 315.15 18,653.20 ± 760.84 [0.02] (14095–23,500)

(122.21–256.31) 186.88 172.61 ± 13.53 (117.83–232.61) 163.75 3.84 ± 0.21 [20.97] (2.72–4.56) 4.12 3.63 ± 0.33 (2.62–5.17) 3.35 371.97 ± 106.64 (105.06–912.80) 184.81 16,330.23 ± 1367.45 [0.02] (10,569.36–21,312.50)

40.58 ± 5.22 (28.4–58.22) 34.94 400.74 ± 18.23 [0.10] (332.81–448.84) 403.71 438.64 ± 23.46 (340.62–490.96) 447.05 1.01 ± 0.05 [40.18] (0.85–1.14) 1.03 1.13 ± 0.06 (0.87–1.25) 1.16 68.90 ± 4.26 (56.09–86.70) 67.56 184.72 ± 20.74 [0.37] (112.85–239.49) 186.58 216.31 ± 44.01 (118.83–353.49) 166.76 4.12 ± 0.46 [16.72] (2.51–5.32) 4.20 4.81 ± 0.98 (2.64–7.86) 3.71 493.44 ± 182.18 (95.85–1277.28) 342.27 15,832.44 ± 1621.93 [0.04] (8977.70–19,371.54)

477.50 ± 48 [0.02] (348.75–799.38) 503.76 474.75 ± 1035.64 (327.50–705.50) 478.82 8.37 ± 0.56 [1.35] (6.39–11.62) 8.49 5.31 ± 0.74 (3.31–10.41) 6.13 58.84 ± 4.61 (40.25–84.24) 57.6 114.38 ± 10.23 [0.51] (76.75–172) 118.13 111.75 ± 7.16 (87.25–148) 114.23 5.34 ± 1.83 [11.02] (1.55–15.13) 7.38 3.71 ± 1.35 (1.42–10.90) 5.66 275.46 ± 75.85 (92.95–790.8) 166.8 19,077.78 ± 943.55 [0.01] (15,757.50–24,125)

543.15 ± 37.05 [0.08] (456.32–639.42) 566.98 575.51 ± 45.13 (477.73–722.90) 546.97 1.38 ± 0.09 [31.83] (1.16–1.63) 1.44 1.47 ± 0.11 (1.22–1.84) 1.39 76.25 ± 8.83 (49.51–95.46) 87.28 221.92 ± 16.31 [0.34] (168.56–250.72) 240.92 204.37 ± 7.49 (189.36–232.14) 196.89 4.87 ± 0.41 [15.66] (3.45–5.57) 5.35 4.53 ± 0.18 (4.12–5.16) 4.38 736.83 ± 212.74 (238.46–1318.64) 567.39 17,741.44 ± 1684.65 [0.04] (12,081.95–22,337.20)

V. Kokkoris et al. / Science of the Total Environment 685 (2019) 280–296


Table 3 (continued) Metal


Coprinus comatus (n = 9)

(mg kg−1 dw)

16–30 cm soil total 0–15 cm soil available 16–30 cm soil available Ni


0–15 cm soil total 16–30 cm soil total 0–15 cm soil available 16–30 cm soil available Cr

Species (no. of samples examined)


0–15 cm soil total 16–30 cm soil total 0–30 cm soil available

19,077.78 19,925 ± 820.45 (15195–22,175) 19,119.72 2.61 ± 1.23 [105.54] (0.73–9.77) 4.20 2.08 ± 1.34 (1.16–11.31) 4.14 13.28 ± 0.45 (11.4–15.6) 13.15 92.88 ± 4.69 [0.14] (75.08–122.22) 93.94 95.90 ± 3.23 (80.05–110.20) 95.3 1.37 ± 0.18 [9.69] (0.70–2.53) 1.40 0.99 ± 0.23 (0.59–2.80) 1.20 0.43 ± 0.43 (0–3.9) 0 51.00 ± 4.46 [0.01] (42.88–81.70) 55.11 60.80 ± 3.6 (43–80.55) 58.39 N.A.

Psathyrella candolleana (n = 14)

Volvopluteus gloiocephalus (n = 15)

Agaricus bisporus (n = 9)

Agaricus bitorquis (n = 6)

Agaricus gennadii (n = 5)

20,192.50 17,625 16,044.51 16,599.68 17,369.22 21,125.18 ± 1484.22 19,182 ± 633.39 16,042.01 ± 1724.43 15,825.78 ± 1569.80 22,608.55 ± 2432.12 (15125–35,262.50) (14,502.50–22,357.50) (10,355.97–27,312.50) (10,217.29–19,487.9) (15,118.58–29,211.48) 20,803.75 19,507.5 15,626.53 16,702.46 23,744.86 5.65 ± 1.16 [61.05] 4.74 ± 1.18 [81.28] 1.29 ± 0.17 [288.19] 1.53 ± 0.17 [322.51] 1.76 ± 0.16 [418.65] (1.13–17.97) (0.81–17.48) (0.47–2.04) (0.90–1.94) (1.21–2.23) 3.87 3.06 1.12 1.59 1.74 3.66 ± 0.60 4.24 ± 1.10 1.21 ± 0.10 1.56 ± 0.17 2.22 ± 0.26 (1.63–10.57) (0.87–15.38) (0.80–1.64) (1.02–1.95) (1.51–2.92) 3.29 2.25 1.21 1.67 2.37 14.55 ± 0.57 16.92 ± 2.79 9.13 ± 0.95 13.41 ± 4.43 10.93 ± 1.47 (11.60–18.88) (11.20–54.92) (6.70–14.62) (7.08–35.29) (7.60–16.18) 14.50 13.4 7.86 9.05 9.61 90.97 ± 4.21 [0.16] 79.51 ± 4.65 [0.21] 143.61 ± 58.02 [0.06] 96.14 ± 3.90 [0.14] 102.23 ± 16.79 [0.11] (51.63–113.40) (51.58–119.62) (69.23–607.25) (85.12–105.23) (51.77–142.19) 93.37 80.95 87.15 98.61 90.89 90.14 ± 3.99 77.92 ± 3.56 136.27 ± 53.87 97.48 ± 3.11 110.43 ± 7.28 (51.43–105.66) (47.28–101.98) (57.02–564.9) (85.68–104.79) (90.90–135.38) 92.70 80.65 85.95 99.15 110.66 1.44 ± 0.11 [10.10] 1.06 ± 0.07 [15.96] 1.79 ± 0.79 [5.10] 1.50 ± 0.38 [8.94] 2.55 ± 0.58 [4.29] (0.95–2.24) (0.56–1.63) (0.41–8.04) (0.59–3.18) (1.18–4.61) 1.30 1.07 0.93 1.33 2.56 1.27 ± 0.11 0.91 ± 0.08 1.29 ± 0.43 1.19 ± 0.25 1.98 ± 0.42 (0.81–1.96) (0.47–1.79) (0.64–4.72) (0.53–2.36) (0.69–3.12) 1.21 0.85 0.87 1.12 1.77 0.68 ± 0.37 0.89 ± 0.42 0 0.69 ± 0.69 0.82 ± 0.82 (0–3.25) (0–4.90) 0 (0–4.12) (0–4.10) 0 0 0 0 0 67.63 ± 7.20 [0.01] 58.53 ± 5.53 [0.02] 65.22 ± 11.66 [0.00] 59.32 ± 10.20 [0.01] 68.82 ± 17.21 [0.01] (34.73–151.80) (29–105.03) (28.92–127.84) (32.64–101.66) (29.64–11.38) 62.78 63.4 71.95 60.38 60.06 64.84 ± 3.65 56.92 ± 4.43 57.28 ± 9.57 58.72 ± 8.76 84.70 ± 15.62 (39.95–88.53) (24.98–80.65) (26.80–100.85) (32.71–91.92) (31.99–115.52) 66.43 58.53 55.29 62.03 102.28 N.A.

of the two metal forms, while for Mn the respective correlations were also positive although not significantly. The results also revealed that certain soils, especially those found in the proximity of roads with heavy traffic, showed high values (above intervention levels) of total metal concentrations which are indicative of pertinent pollution. However, no significant correlations were observed between the content of heavy metals in mushrooms and the proximity of their collection sites to main roads. 3.2. Metal concentrations in mushrooms Measurements of metal concentrations in mushrooms are summarized in Table 3 and graphically depicted in the box-plots (Fig. 2; Supplementary material, Fig. S1). The principal component scores (PC1 and PC2) accounted for 50.5% of total variance (Supplementary material, Fig. S2). A. bitorquis and A. gennadii were differentiated based on the heavy metal content of their fruit-bodies compared to C. comatus and P. candolleana, but not from A. bisporus and V. gloiocephalus. More specifically: 3.2.1. Chromium The results of the present work revealed low concentrations of Cr (several of them being below the instrument's detection limit), while maximum values did not exceed the 5 mg kg−1 threshold ranging from 3.25 to 4.90 mg kg−1 for the five out of six species examined (no Cr was detected in A. bisporus). Such measurements for wild mushrooms are in accordance with the outcome of most previous studies





(Cr content: 0.5 to 5 mg kg−1; Ouzouni et al., 2009; Kalač, 2010; Širić et al., 2016; Bosiacki et al., 2018). However, there are also reports referring to values exceeding 10 mg kg−1 even for fruit-bodies collected from allegedly not-polluted areas (Isildak et al., 2007; Ouzouni et al., 2007) including samples of A. bisporus and C. comatus (Zhu et al., 2011).

3.2.2. Copper Cu content of mushroom samples examined in this study ranged from 5.57 to 165.66 mg kg−1, while the median values were 52.30, 47.39, 35.20, 51.34, 51.19 and 8.33 mg kg−1 for C. comatus, P. candolleana, V. gloiocephalus, A. bisporus, A. bitorquis and A. gennadii, respectively. A rather wide variation of Cu concentration in edible mushrooms was reported in the past; in most cases, values are lower than 100 mg kg−1 (Falandysz et al., 2011; Giannaccini et al., 2012; Zhang et al., 2013; Borovička et al., 2019) including measurements on samples of C. comatus, V. gloiocephalus and A. bisporus (Zhu et al., 2011; Sarikurkcu et al., 2012; Bosiacki et al., 2018). However, there are also studies referring to concentrations exceeding 200 mg kg−1 in mushrooms collected from rural areas (Alonso et al., 2003; Svoboda and Chrastný, 2008; Jarzyńska et al., 2011) reaching up to 500 mg kg−1 in fruit-bodies found around copper smelters (Svoboda et al., 2000; Collin-Hansen et al., 2002). Relatively large sets of data deriving from the comparative examination of Cu accumulation by Boletus edulis, Macrolepiota procera and other species indicated that its uptake varies among different soil substrates and depends on the fungus (Falandysz and Borovička, 2013).

288 V. Kokkoris et al. / Science of the Total Environment 685 (2019) 280–296 Fig. 2. Concentration of each metal (mg kg−1) in the fruit-bodies of six species examined, i.e. AB: Agaricus bisporus, AG: A. gennadii, AT: A. bitorquis, CC: Coprinus comatus, PC: Psathyrella candolleana and VG: Volvopluteus gloiocephalus. Box-plots show the third quartile and first quartile (box edges), median (middle line), range of data (whiskers) and data outliers (circles).

V. Kokkoris et al. / Science of the Total Environment 685 (2019) 280–296

3.2.3. Iron The results of this study revealed a wide variability in the Fe content of the mushroom samples examined ranging from 81.85 to 1318.64 mg kg−1, while the median values were 166.80, 323.93, 315.15, 184.81, 342.27 and 567.39 mg kg−1 for C. comatus, P. candolleana, V. gloiocephalus, A. bisporus, A. bitorquis and A. gennadii respectively. Similarly, values for iron concentrations demonstrated a high variation among wild mushrooms examined in past studies. They were rarely reported to be lower than 50 mg kg−1 (Borovička and Řanda, 2007) but they commonly exceeded 100 mg kg−1 (Isildak et al., 2004; Rudawska and Leski, 2005; Zhu et al., 2011; Sarikurkcu et al., 2012; Sun et al., 2017). 3.2.4. Lead The Pb content of the studied samples ranged from 0 to 16.81 mg kg−1, while the median values were 6.70, 7.46, 9.00, 3.61, 2.31 and 4.88 mg kg−1 for C. comatus, P. candolleana, V. gloiocephalus, A. bisporus, A. bitorquis and A. gennadii respectively. Past studies on numerous mushroom species collected from unpolluted areas evidenced lower Pb concentrations which in most cases did not exceed 6 mg kg−1 (Kalač, 2010; Falandysz and Borovička, 2013; Sun et al., 2017; Falandysz, 2018) including measurements on C. comatus, V. gloiocephalus and A. bisporus (Isildak et al., 2007; Zhu et al., 2011; Sarikurkcu et al., 2012). Reported exceptions originated either from urban areas with concentration values in the range of 10–15 mg kg−1 (Garcia et al., 1998; Schlecht and Säumel, 2015) or from heavily polluted mining sites with values of up to 270 mg kg−1 (Kojta et al., 2012; Borovička et al., 2019). 3.2.5. Manganese The Mn content of the mushroom samples examined in this study showed a high variability ranging from 3.75 to 59.14 mg kg−1, while the median values were 7.50, 10.28, 25.28, 32.41, 34.94 and 38.72 mg kg−1 for C. comatus, P. candolleana, V. gloiocephalus, A. bisporus, A. bitorquis and A. gennadii respectively. These values are in accordance with the outcome of most studies reporting concentrations usually lower than 50 mg kg−1 from unpolluted areas (Rudawska and Leski, 2005; Isildak et al., 2007; Ouzouni et al., 2007, 2009). 3.2.6. Nickel The Ni content of the studied mushrooms ranged from 6.70 to 54.92 mg kg−1, while the median values were 13.15, 14.50, 13.40, 7.86, 9.05 and 9.61 mg kg−1 for C. comatus, P. candolleana, V. gloiocephalus, A. bisporus, A. bitorquis and A. gennadii respectively. Such concentrations are significantly higher than most of those reported in pertinent literature (as reviewed by Kalač, 2010, and Širić et al., 2016) which include measurements on C. comatus, V. gloiocephalus and A. bisporus (Isildak et al., 2007; Zhu et al., 2011; Sarikurkcu et al., 2012; Bosiacki et al., 2018). 3.2.7. Zinc The Zn content of the mushroom samples examined in this study ranged from 40.25 to 155.27 mg kg−1, while the median values were 57.60, 86.98, 79.48, 71.05, 67.56 and 87.28 mg kg−1 for C. comatus, P. candolleana, V. gloiocephalus, A. bisporus, A. bitorquis and A. gennadii respectively. Similar values were reported in several other studies examining mushroom samples from non-polluted areas including fruitbodies of C. comatus, V. gloiocephalus and A. bisporus (Rudawska and Leski, 2005; Isildak et al., 2007; Zhu et al., 2011; Giannaccini et al., 2012; Bosiacki et al., 2018; Borovička et al., 2019). However, values exceeding 150 mg kg−1 were also recorded for certain edible species, e.g. Agaricus campestris, A. macrosporus, Calvatia utriformis, Leccinum scabrum, Lycoperdon perlatum etc. (Alonso et al., 2003; Borovička and Řanda, 2007; Falandysz et al., 2011), these being considerably higher in mushrooms sampled near a zinc smelter (Collin-Hansen et al., 2002).


3.2.8. Potassium, sodium and phosphorus P. candolleana, V. gloiocephalus and Agaricus spp. accumulated high amounts of K (median values 83.12, 41.75 and 34.99 g kg−1 respectively), whereas C. comatus showed notably lower concentrations (median value 9.95 g kg−1) (Supplementary Material, Table S1). Similarly, mushrooms of P. candolleana, V. gloiocephalus and Agaricus spp. presented higher concentrations of phosphorus than C. comatus (median values of 9.86, 12.96 and 9.29 g kg−1 as opposed to 6.59 g kg−1). As regards Na, C. comatus demonstrated slightly higher concentrations (median value 20.63 g kg−1) when compared to P. candolleana and V. gloiocephalus (median values of 16.53 and 16.63 g kg−1 respectively), while Agaricus spp. showed the lowest concentrations (median value 10.27 g kg−1). Once again, the results indicated that uptake of nutrients differs significantly among fungal species even if concentrations of K, Na and P did not present high variations in the soils of the studied area. 3.3. Correlations and relationships among parameters examined – metals bioavailability and uptake by mushrooms Concentrations of metals in mushrooms were established and evaluated vis-à-vis the concentrations of available and total metals in the respective soil substrates (Table 3). Common methods employed for metal extraction from soils (through the use of nitric acid, nitric and sulphuric acids or aqua regia) target almost all metal pools in soils and not exclusively the bio-available fraction; hence they might not arrive at detecting/evidencing correlations between metals concentration in mushroom and soils. In contrast, extraction via DTPA assesses the potentially available pool of metals in soils (Feng et al., 2005; Coelho et al., 2018) and could provide an interesting insight in the pertinent bioaccumulation process. In fact, the results of this study demonstrated statistically significant correlations between available metal concentrations in soils versus their respective content in the fruit-bodies of the species examined (Fig. 3). Hence, Mn concentrations in A. bisporus and C. comatus mushrooms are significantly (negatively) correlated with their bioavailable concentrations in both soil layers (r = −0.64 at p b 0.001, and r = −0.78 at p b 0.05 respectively, in both 0–15 and 16–30 cm horizons), whereas bioavailable Ni was positively correlated with its content in V. gloiocephalus fruit-bodies (r = 0.84 at p b 0.05, in both 0–15 and 16–30 cm horizons). In addition, bioavailable Zn and Cu were found to be significantly correlated with their content in P. candolleana and V. gloiocephalus respectively (r = 0.60 and r = 0.55 at p b 0.05, for the upper soil layer). In contrast, no significant correlations were detected between heavy metals total concentrations in soils versus their respective content in mushrooms. Besides direct comparisons between contents of the same metal in mushrooms and in their soil substrates, the effect of different elements concentrations in soil versus their accumulation in fruitbodies was also examined. However, no significant correlations were detected in any combination tested between heavy metals. In contrast, K, Na and P presented notable correlations between their available concentrations in soils versus the content of several metals in fruit-bodies (Fig. 3). Among them, Na concentration in the upper soil layer was negatively correlated with Pb content in C. comatus and P. candolleana (r = −0.83 at p b 0.01 in the upper soil layer for the former species, and r = −0.73 and r = −0.76 at p b 0.01 in the upper and lower soil layer respectively for the latter species). Similarly, K showed negative correlation with Pb content in C. comatus and P. candolleana (r = −0.73 and r = −0.57 at p b 0.01 in the upper and lower soil layer respectively). On the other hand, concentrations in the upper and lower soil layers of both K and Na exhibited significant correlations with other metals content in V. gloiocephalus fruit-bodies, i.e. K was positively associated with Cu whereas Na was negatively correlated with Fe and Ni (Fig. 3). As regards P, it showed positive correlations with Cu (r = 0.80 and r = 0.85 at p b 0.01 in the upper and lower soil layer, respectively) and Pb (r = 0.84 at p b 0.01 in the lower soil layer only) in C. comatus mushrooms, whereas it

290 V. Kokkoris et al. / Science of the Total Environment 685 (2019) 280–296 Fig. 3. Interactions/correlations between metals content in the fruit-bodies of (a) Agaricus bisporus (b) Coprinus comatus, (c) Psathyrella candolleana, (d) Volvopluteus gloiocephalus and available concentrations of various elements in the two soil horizons examined (0–15 cm and 16–30 cm). Black and red arrow lines represent positive and negative correlations respectively. Levels of statistical significance are depicted as follows: p b 0.05 (*, dotted line); p b 0.01 (**, solid/thin line); p b 0.001 (***, solid/thick line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

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presented negative correlations with Cu (r = −0.59 at p b 0.05 in the upper soil layer) in P. candolleana. Of significant interest is the distribution of soil substrates in respect to their texture type and the pertinent preference exhibited by each mushroom species examined (Fig. 4). C. comatus was found to grow in soils with the narrowest spectrum of texture types (loam to clay loam), V. gloiocephalus was sampled from sandy loam to clay loam soils, whereas P. candolleana presented the highest adaptability on a wide range of soil types. On the other hand, Agaricus spp. presented distinct preference towards sandy loam (A. bisporus), sandy clay loam (A. bitorquis) and clay (A. gennadii) soils. Of importance was also how soil characteristics affected the availability of metals and subsequently the ability of mushrooms to absorb them. Results demonstrated that the effect of soil texture on metal accumulation was species dependent since high correlations were mainly found for V. gloiocephalus (and to a lesser extent for C. comatus), whereas no effect of this type was detected for Agaricus spp. and P. candolleana. In the former case, sand content was found to be negatively (albeit not always at a statistically significant level) related to the uptake of certain metals by fungi, whereas clay and silt content in soils seem to positively affect bioaccumulation of some elements. More specifically, Cu and Zn concentrations in V. gloiocephalus were found to be highly correlated to soil mechanical composition: r = −0.78, r = 0.83 and r = 0.74 for Cu, and r = −0.81, r = 0.77 and r = 0.70 at p b 0.05 for Zn, for sand, clay and silt respectively in the upper soil layer (similarly, high values were also obtained for the lower soil layer). In addition, clay content was positively correlated to Fe and Ni accumulation by C. comatus (r = 0.60 and r = 0.64 at p b 0.05 and p b 0.01 respectively for the lower soil layer), whereas sand content was negatively correlated with Fe absorption by the same species (r = −0.61 at p b 0.05 for the upper soil layer). The observed significant positive correlations between the clay (and silt) content in soils versus metals concentration in mushrooms could be attributed to the presence of negatively charged sites on clay surfaces (and on clay particles that partly cover silt) which favor metal retention in exchangeable forms. Inorganic soil colloids possess sites for metals retention. Among them, metal oxides (and especially Fe oxides) sequester metals introduced in the soil and regulate/affect their availability (Kalyvas et al., 2018 and references therein). It is possible therefore, that fungal exudates may promote the release of the metals mainly associated with amorphous oxides, increasing their availability and favoring their increased uptake by the hyphae. On the other hand, eqCaCO3 did not seem to exert a consistent and statistically significant effect on the accumulation of metals by mushrooms, while soils pH values demonstrated different correlations with various metals for each species studied, i.e., negative for Pb and Zn in C. comatus, and for Mn and Cu in P. candolleana, and positive for Ni in V. gloiocephalus. Organic matter content presented a notable influence only in the case of Fe and Ni uptake by P. candolleana (r = −0.50 and r = −0.54 at p b 0.05 respectively for the upper soil layer). As previously shown, metal concentration in soil from different depths was correlated to the element content in mushrooms. The deposition of heavy metals from anthropogenic activities enriches the soil (the upper layer presents higher content in organic matter while the soil beneath is mainly associated to mineral horizons) and depending on the soil characteristics and the effects of biotic and abiotic factors, they become available to soil biota. Uptake of each metal from this pool is performed in a different manner by each fungal species (e.g. through chelating/binding agents of protein nature, or specific functional sulphydryl-, carboxylic- and/or amino-groups; Michelot et al., 1998) and is under the influence of various factors, e.g. environmental conditions, soil properties, and/or ability of the mycelium to grow deeper into the soil. Last, some interesting observations were made when individual metal concentrations in mushrooms were compared with each other (Fig. 5). All species showed highly positive correlations between Mn


and Fe (e.g. r = 0.60, r = 0.93, r = 0.94 and r = 0.86 at p b 0.001 for A. bisporus, C. comatus, P. candolleana and V. gloiocephalus respectively), and Fe with Ni (e.g. r = 1, r = 1, r = 0.93 and r = 0.77 at p b 0.01 for Α. bitorquis, A. gennadii, C. comatus and P. candolleana respectively). In addition, Mn was also correlated with Ni for Α. bitorquis, C. comatus and P. candolleana (r = 1, r = 0.96 and r = 0.63 at p b 0.01, p b 0.001 and p b 0.05 respectively), and Cu with Zn for P. candolleana and V. gloiocephalus (r = 0.57 and r = 0.75 at p b 0.05 and p b 0.01 respectively). Of interest were also the positive correlations of Zn vs. Pb in C. comatus (r = 0.68, at p b 0.05), Pb vs. Ni and Fe in A. bitorquis and V. gloiocephalus (r = 0.89 and r = 0.62 at p b 0.05 respectively) as well for Cr vs. Fe in V. gloiocephalus (r = 0.58 at p b 0.01). Noteworthy were the negative correlations found for Cu with Ni and Pb with Cr in P. candolleana (r = −0.72 and r = −0.53 at p b 0.01 and p b 0.05 respectively). In addition, no correlations were established whatsoever for three metals (Cu, Cr and Zn) in Agaricus spp. Most of the interactions between metals content in fruit-bodies seem to be species-specific except for Ni\\Fe and Mn\\Fe, which are positively correlated for all mushrooms suggesting a synergistic uptake mechanism for these essential elements. 3.4. Evaluation of metals accumulation by mushrooms The ability of the fungus to accumulate metals from its growth substrate is commonly evaluated through the use of the bioconcentration factor (BCF), i.e., the ratio of the metal content in the fruit-body to its content in underlying substrate. For the purposes of this study, BCFs were calculated by taking into account the total or the available metals concentration in the top 15 cm soil layer (Table 3). Overaccumulation was established only when the available fractions of Cu, Mn, Zn and Ni in soil were considered (bioconcentration factors: 1 to 41). Cu was the only one among the elements examined to show relatively high accumulation in mushrooms in respect to its total content in four out of six mushroom species studied, i.e. C. comatus: 1.79, P. candolleana: 1.05, A. bisporus and A. bitorquis: 0.98; such values are similar to those previously obtained for other mushrooms (Sun et al., 2017). Especially as regards Zn, previously reported BCFs were usually quite higher than 1 (Alonso et al., 2003; Sun et al., 2017; Falandysz et al., 2018) whereas in the present study the respective values for different mushroom species were quite inferior (BCF: 0.34–0.51); it was only when the available Zn was taken into account that BCFs reached values of 5 to 21. Bioconcentration values for Ni were significantly lower than 1 (0.1–0.2) only when calculations were made on the basis of the total metal content; this being in agreement with literature findings (Aloupi et al., 2012; Gucia et al., 2012; Garcia et al., 2013; Drewnowska and Falandysz, 2015; Širić et al., 2016). In contrast, when available fraction of Ni in soil was considered, then BCF values ranging from 4 to 16 were obtained. Bioconcentration values for Cr were found to be particularly low (BCF: 0.01–0.02) and this is congruence with pertinent literature data reporting BCF b 0.2 (Garcia et al., 2013; Drewnowska and Falandysz, 2015; Širić et al., 2016). As regards Fe, BCF values are very low (0.01–0.04) in accordance to former studies (Malinowska et al., 2004; Sun et al., 2017); however, when BCFs were calculated on the basis of the available Fe fraction in soil, then all species demonstrated hyperaccumulation of this metal reaching values of 61 to 419. For Pb, bioconcentration did not exceed the value of 1 for both the total and available fractions in soil, which is in accordance with the low BCF values reported in other mushrooms for this particular element (García et al., 2009; Drewnowska and Falandysz, 2015; Širić et al., 2016; Sun et al., 2017). Of interest was the fact that all three Agaricus spp. demonstrated a distinct behavior (possibly due to intrinsic or “organism-dependent” factors) in accumulating certain metals (i.e., Mn, Fe, and to lesser extent Zn) when compared to the other species examined as evidenced by the markedly higher BCFs they presented when available metal concentration values were taken into account. On the other hand, the lower efficiency in accumulating certain heavy metals

292 V. Kokkoris et al. / Science of the Total Environment 685 (2019) 280–296 Fig. 4. Distribution of substrates (in respect to different soil types as these are depicted on the soil texture triangle) from which fruit-bodies of the following species were sampled: Agaricus bisporus (yellow dots), A. bitorquis (grey dots), A. gennadii (orange dots), Coprinus comatus (red dots), Psathyrella candolleana (green dots) and Volvopluteus gloiocephalus (blue dots). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

V. Kokkoris et al. / Science of the Total Environment 685 (2019) 280–296 Fig. 5. Spearman's correlations between metals concentrations measured in the fruit-bodies of six species: (a) Agaricus gennadii, (b) A. bisporus, (c) A. bitorquis, (d) Coprinus comatus, (e) Psathyrella candolleana, (f) Volvopluteus gloiocephalus. Black arrows represent positive correlations and red arrows represent negative correlations. Metals not showing any statistically significant correlations are presented within light-coloured circles. Levels of statistical significance are depicted as follows: p b 0.05 (*, dotted line); p b 0.01 (**, solid/thin line); p b 0.001 (***, solid/thick line). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)



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(e.g. Pb and Cr) could be attributed to their possible toxic effects to the fungus (García et al., 2009). Bioexclusion of heavy metals is considered as a defense mechanism protecting fungi against excessive concentrations in soil substrates although several species were reported to efficiently accumulate metallic elements and metalloids in their fruitbodies even in areas without any particular human activities (Michelot et al., 1998; Isildak et al., 2007). In general, from the rather low number of studies (including the present one, i.e. the first performed within a heavily urbanized area) which assessed the content of metals in both fruit-bodies and their substrates, it could be deduced that bioaccumulation varies considerably depending on the element and the fungal species. Furthermore, and in the light of the findings of this work, previous conclusions on whether mushrooms are bioaccumulators or bioexclusors for certain metals (e.g. Agaricus campestris, Clitocybe inversa, C. nebularis, Macrolepiota procera, Armillaria mellea, Boletus aestivalis, B. edulis, Lactarius deterrimus, Tricholoma portentosum and T. terreum, all considered as Ni bioexclusors; Širić et al., 2016) should be revised to include estimations related with the available fraction of each element in soil and on how environmental factors such as soil properties and atmospheric depositions (Blake and Goulding, 2002; Xu et al., 2017), affect availability of metals and their accessibility to mushrooms. 3.5. Potential impact from the consumption of mushrooms on humans For determining whether the edible mushrooms collected in the frame of this study from the Athens urban area are safe or not for human consumption, the regulations of the European Commission, when available (, the tolerable upper intake levels (i.e., the highest level of daily nutrient intake that is likely to pose no risk of adverse health effects for almost all individuals, UL) and/or the recommended dietary allowance (RDA) for adults were considered for each metal and mushroom species studied. For five out of the six mushroom species examined and for the majority of the samples included in the analysis (i.e., 5 out of 9 for A. bisporus, 3 out of 5 for A. gennadii, 7 out of 9 for C. comatus, 8 out of 14 for P. candolleana and 12 out of 15 for V. gloiocephalus), Pb content surpassed the maximum limit of 0.3 mg kg−1 fresh weight (or 3 mg kg−1 dry weight assuming a 90% moisture content) set by the EC Regulation No 629/2008 (European Commission, 2008). Exceeding these limit values is not unusual for Pb in wild mushrooms since several studies reported similar results (Gucia et al., 2012; Árvay et al., 2015; Schlecht and Säumel, 2015; Stefanović et al., 2016). On the other hand, if the provisional tolerable weekly intake (PTWI) for Pb (i.e. 0.025 mg kg−1 bodyweight week−1) is taken into account (JECFA, 2010), then for a person weighing 70 kg, this corresponds to 1.75 g (or ca. 5 servings per week; one serving corresponds to the consumption of 300 g f.w. or of approx. 30 g d.w.) which could pose a threat for human health if consumed regularly. As regards Cu, RDA for adults is 0.91 mg day−1 (0.013 mg kg−1 day−1) (Agency for Toxic Substances and Disease Registry (ATSDR), U.S. Department of Health and Human Services; meaning that one serving of any one of the six edible mushroom species collected in the area of study could confer over 1 mg Cu, which is higher than the respective RDA (and quite higher in the cases of the maximum values recorded in two A. bisporus and A. bitorquis specimens). Still, Cu concentrations in the mushrooms of this study are lower than the UL level of 10 mg day−1. Although no RDA has been established for Ni, its common daily intake by adults is estimated at 0.1–0.14 mg day−1 (ATSDR; https:// Therefore, consumption of a serving of the edible mushrooms examined in this work corresponds to Ni intake which is higher than the value quoted above (up to 0.45 mg Ni). Zinc intake from food varies highly and depends on several factors (e.g. on interactions with other metals like antagonism between Zn\\Cd

and Zn\\Cu) while increased levels of Ca and Mg could inhibit its availability (Kabata-Pendias and Pendias, 1992). RDA for Zn has been established at 11 mg day−1 for men and 8 mg day−1 for women (ATSDR; Consumption of one daily serving of any of the studied edible mushrooms provides lower amount of this metal in respect to the values quoted (on average b3 mg day−1 Zn). RDA for Fe in adults varies from 10 to 18 mg day−1 and might be higher for pregnant women and adolescents, while UL is established at 45 mg day−1 (Institute of Medicine (IOM) – Food and Nutrition Board, 2001). Therefore, the amounts of Fe present in a daily serving of the edible mushrooms of this study are similar to the RDA value (they correspond to ca. 6–18 mg day−1). Since no RDAs have been established for Cr, Adequate Intakes (AIs) were developed instead, on the basis of average intakes of Cr from food which range from 0.020 to 0.035 μg day−1 (Institute of Medicine (IOM) – Food and Nutrition Board, 2001). These values are much lower to those that correspond to a daily serving of mushrooms collected from the area under study (up to 0.15 mg day−1). 4. Conclusions The mushroom species analyzed in this study showed differences in the way they accumulate heavy metals and macronutrients, i.e. V. gloiocephalus exhibited the highest Pb and Ni content, A. bisporus and A. bitorquis the highest Cu content, while the two latter alongside with A. gennadii presented the highest Mn content among all mushrooms examined, and P. candolleana demonstrated the highest Zn concentration. Soil characteristics affect metal mobility and availability, and could have a notable effect on their final concentrations in mushrooms; this was the case for C. comatus and V. gloiocephalus, whereas P. candolleana and Agaricus spp. were less affected by soil metal content and properties indicating that the final concentration of metals in the fruit-bodies highly depends on the mushroom species. Statistical analysis of metals concentration in mushrooms and their respective soil substrates evidenced significant correlations of the former with the pool of bioavailable elements in soil (and in contrast to their total content in soil). In addition, soil characteristics (e.g. texture, pH, organic matter, content in macronutrients) influenced the amount of metals detected in mushrooms. In general, the final concentration of metals in the fruit-bodies seems to depend on one or more of the following factors and their interactions: (a) the mushroom species, (b) the pool of available metals concentrations in soil, (c) the soil properties, (d) the relative abundance of elements in soil and their antagonistic and/or synergistic effects, and (e) the relative concentration of metals in fruit-bodies (inasmuch as some metals, different for each species, interact with each other in enhancing/obstructing absorption of minerals from soil). Although most soil samples did not appear to be particularly rich in heavy metals, consumption of urban mushrooms should be exercised with great care since their content in Pb and Cu exceeded the maximum tolerable daily intake and RDA, respectively. CRediT authorship contribution statement Vasilis Kokkoris: Conceptualization, Methodology, Software, Validation, Formal analysis, Investigation, Data curation, Writing - original draft, Writing - review & editing, Visualization. Ioannis Massas: Conceptualization, Methodology, Validation, Investigation, Resources, Data curation, Writing - original draft, Writing - review & editing, Supervision, Project administration, Funding acquisition. Elias Polemis: Methodology, Investigation, Writing - original draft. Georgios Koutrotsios: Methodology, Software, Investigation, Writing - original draft. Georgios I. Zervakis: Conceptualization, Methodology, Validation, Formal analysis, Investigation, Resources, Data curation, Writing - original draft, Writing - review & editing, Visualization, Supervision, Project administration, Funding acquisition.

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Acknowledgements We would like to thank Dr. O. Kairis for the preparation of Fig. 1. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Declaration of Competing Interest The authors declare that they have no conflict of interest. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi. org/10.1016/j.scitotenv.2019.05.447.

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