Rare Earth Elements in Boletus edulis (King Bolete) Mushrooms from Lowland and Montane Areas in Poland

Mining/exploitation and commercial applications of the rare-earth elements (REEs: La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu) in the past 3 decades have raised concerns about their emissions to the environment, possible accumulation in food webs, and occupational/environmental health effects. The occurrence and distribution of REEs Y and Sc in the fruitbodies of Boletus edulis collected from geographically diverse regions in Poland were studied in 14 composite samples that were derived from 261 whole fruiting bodies. Individual REE median concentrations ranged from 0.4–95 µg kg−1 dry weight (dw). The summed REE concentrations varied widely, with a median value of 310 µg kg−1 dw and a range of 87 to 758 µg kg−1. The Sc and Y median concentrations (dw) were 35 and 42 µg kg−1, respectively. Ce, La, and Nd, with median values of 95, 51, and 32 µg kg−1, respectively, showed the highest occurrence. B. edulis collected from a forested area formerly used as a military shooting range—possibly a historically contaminated site—had an elevated summed REE content of 1796 µg kg−1. REE concentrations were generally low in Polish King Bolete. Dietary intake from a mushroom meal was negligible, posing no health risk to consumers.


Introduction
The term rare-earth elements (REEs) includes La, Ce, Pr, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu-all of the lanthanides series [1]. Scandium (Sc) and yttrium (Y) are also studied in foods and environmental materials and considered together with the REEs [2][3][4]. The REEs, Sc and Y occur in soil at relatively low concentrations and their mineral deposits worldwide are limited, thus they are considered critical metals [5,6]. Nowadays, REEs are a highly sought after resource for industrial technology and numerous other applications [7]. Mining and processing of the REE ores like that of some other metallic elements often result in pollution of underground waters, the pedosphere, and atmosphere as well as in elevated levels in vegetation at least at the local or regional level [8][9][10].
In an early study by Ichihashi and co-workers (1992), the herb pokeweed (Phytolacca americana) and two species of ferns (Dicranopteris dichotoma and Athyrium yokoscence) were observed to be good accumulators of REE Sc and Y compared to other species investigated, while the species Ph. americana closely reflected their pattern in the soils [11]. Concentration of REEs in plants can be higher than is typical if they grew in polluted soil [3,6,12].
From the food, environmental, and toxicological point of view, the intentional or unintentional production, release, exhaust, or other emissions of REEs pose a risk to the health of humans and other biota. The particle size of the emission/release may determine the deposition/exposure, and this may be more harmful if they are nanoparticles [13]. Apart from  Mushrooms used in this study were specimens from a repository [36]. Before storage in dry and clean conditions, the fungal materials were properly cleaned, dried, and ground after collection and packed in tightly closed plastic containers. Mushrooms that are processed and stored in these conditions are considered to be valid materials for the study of minerals and trace elements decades after collection [39]. To gain insight into the distribution of REEs, Sc and Y between the two major morphological parts of the fruiting body, the specimens from some locations were separated into cap (or pileus with skin) and stipe (or stem). Depending on the number collected at each site, between 10 and 45 specimens were pooled into composites by location ( Figure 1; Table 1) [40].
To determine the REEs, Sc and Y, circa 200 mg quantities of finely powdered composites of the fungal materials were mixed with 3 mL solution of ultrapure concentrated nitric acid (HNO 3 , 65%) and 1 mL of ultrapure hydrofluoric acid in a polytetrafluoroethylene (PTFE) tube. Then, the tubes were screw-tightened in a stainless steel jacket and digested in an oven at 150 • C for 78 h. The resulting solutions obtained were evaporated to dryness at 110 • C to remove the excess HF, dissolved in about 1 mL of HNO 3 in a clean sample tube, and diluted to a final volume of 50 mL with ultrapure water. As an internal standard, rhodium (Rh) (10-20 micrograms per litre) was added to each sample prior to the analysis by inductively coupled plasma mass spectrometry (ICP-MS, ELAN DRC-e, PerkinElmer Inc., Woodbridge, ON, Canada) [24,41].
In order to achieve good analytical quality control and quality assurance, blanks and two certified reference materials (CRMs) were examined. Each element was measured 3 times and the values of relative standard deviation (RSD) were within 5% in the samples and the certified values for the CRM. Due to unavailability of a REE CRM for mushrooms, citrus leaf (GBW 10020) and soil (GBW 07405) CRMs produced by the Institute of Geophysical and Geochemical Exploration, China, were used [40,42]. The limit of quantification of the method for individual elements was in the range from 0.1 ng g −1 to 2 ng g −1 . The results obtained for B. edulis were further normalized against REEY patterns in certain shales [25,43].
REE intake through mushroom consumption was estimated using the following equations [44]: where EDI is the estimated daily intake of REEs from mushroom consumption; Cm is the ∑REE in the mushroom (wet weight); CR is the consumption rate [100 g wet (whole) weight for normal consumers and 300 g wet (whole) weight for high level consumers]; and BW is the body weight (60 kg for adults and 30 kg for children). The intake dose of REEs that was found to be damaging to human health was reported to be 100-110 µg kg −1 BW day −1 , which was certificated through a human health study in REEs mining areas and from animal experiment results [3,45,46].

REE Concentration Levels
The range of values for each REE, Sc and Y, determined in B. edulis are listed by morphological part of the fruiting body and place of collection in Table 1. The median values of the individual elements derived for whole fruiting bodies, caps, and stipes in this study were in the range (in µg kg −1 dw), 5.9 to 35 for Sc, 31 to 43 for Y, 33 to 56 for La, 60 to 110 for Ce, 6.4 to 11 for Pr, 22 to 41 for Nd, 3.4 to 6.3 for Sm, 0.6 to 1.1 for Eu, 4.3 to 5.2 for Gd, 0.7 to 1.2 for Tb, 3.8 to 5.5 for Dy, 0.7 to 1.1 for Ho, 2.1 to 3.6 for Er, 0.4 to 0.6 for Tm, 2.0 to 3.5 for Yb, and 0.4 to 0.5 for Lu (Table 1). These median values for the REEs as well as other data obtained for almost all locations in this study agree very well with an earlier report on the concentrations of lanthanides (using sector field mass spectrometry) accumulated in a few B. edulis caps collected at some locations from the northern region of Poland [21].
Macromycetes take up from soil substrata both organics and minerals and accumulate these in the mycelium, and mycorrhizal species also share some nutrients and water with symbiotic plants. They are then gradually translocated to the fruiting bodies based on needs-this is believed to be largely regulated in the case of the macronutrients (K, P, S, Mg) and major micronutrients (Zn, Cu, Ca, Na, Se). The co-accumulation of some other elements alongside those listed above also follows the same physiological pathways, often because of physical and chemical similarities between the elements, though some particular mushroom species show specific ability for accumulation of certain elements, e.g., V (some species need it), Ag, Hg, etc. Despite this, soil geogenic signature and especially anthropogenic pollution (As, Cd, Hg, Pb, Sb, 137 Cs, 90 Sr, etc.) also contribute much to the uptake and accumulation of elements by mushrooms. This is observed in studies showing that mushrooms harvested from significantly polluted sites show higher levels of such elements-above the values typical for their species [20,47].
The stipes of fruiting bodies in the present study usually showed higher concentrations of REEs than the caps and the median values of the sum of the REEs (∑REE) at four sites were 359 and 186 µg kg −1 dw, respectively ( Table 1). The observed higher values of the ∑REE in the stipes compared to the caps of the fruiting bodies of B. edulis can be explained by considering the predilection of these elements to Ca [48]. Stipes in mature fruiting bodies are usually richer in Ca. On the other hand, because of their similar chemical and physical properties, REEs are supposed to be absorbed by organisms (e.g., mushrooms) as a group [49]. Divalent Ca, which readily co-occurs in organisms with REEs, occurs at much higher levels in mushrooms, around three to four orders of magnitude, than REEs. For example, studies have reported mean Ca values in caps of B. edulis at 39,000 ± 20,000 µg kg −1 dw (n = 4), while another study reported median values in the range of 29,000 to 170,000 µg kg −1 dw for caps and from 54,000 to 250,000 µg kg −1 dw for stipes (n = 144) [40,50].
Available results for REEs (Table 1) and Ca levels in the sample sets of B. edulis [21,40,50] indicate a positive correlation in co-occurrence, but observations are too few to examine regression. For example, Ca in B. edulis from the Sudety Mts. site had a median Ca concentration level of 160 mg kg −1 dw in stipes and 83 mg kg −1 dw in caps (id 15; Table 1), and for mushrooms from the Giżycko site, the Ca concentration was 110 mg kg −1 dw in stipes and 91 mg kg −1 dw in caps (id 10; Table 1) [40,50]. Some similarity in the distribution of REEs and Ca was also found in the mushroom Lactarius pubescens [26]. In Macrolepiota procera (Field Parasol or Parasol Mushroom) REE levels correlated with Ca and also with Na and Mg [4].
Macrofungi are rather poor Ca bio-concentrators and this is even worse for REEs. For example, the Ca bioconcentration factor (BCF; otherwise quotient from concentration in mushroom and concentration in soil) for a large collection of B. edulis ranged from 0.08 to 1.5, and for Leccinum scabrum (Rough-stemmed Bolete) from 0.003 to 0.027) [39,51].
The little available data for REEs (such as La and Ce) in mushrooms (Amanita pantherina, Lactarius hatsudake, Russula mariae, Suillus granulatus, and Tricholoma flavovirens) showed BCF values in the range from 0.003 to 0.027 (La) and from 0.0003 to 0.025 (Ce) [52].
Cerium was the most abundant REE determined in B. edulis. Mushrooms collected from the Morąg site in the Warmia region showed an order of magnitude greater quantities of Y, La, Ce, Nd, Sm, Eu, Gd, Tb, Dy, Ho, Er, Tm, Yb, and Lu than specimens from other sites ( Table 1). If we exclude any cross-contamination with incrusted sand particles, no other reason can be attributed to this observation other than to associate it with the legacy of an abandoned military area overgrown with forest near the town of Morąg, where mushrooms were collected. B. edulis (and some other species from the Morąg site) relative to other locations also showed higher concentrations of other metallic elements (Ba, Co, Ga, Ge, Hf, Hg, Li, Nb, Pb, Sr, Ti, U, V, and Zr) [36], though the mushroom Amanita muscaria was not studied for Ba, Ga, Pb, or Sr (and the REE) and Paxillus involutus had only data for Hg [53,54].
Aruguete et al. earlier discussed the possible impact of geochemical background composition on the abundance of REEs in mushrooms. In their study, differences were observed in the uptake and sequestration of La, Ce, and Nd in fruiting bodies by edible Amanita rubescens and Amanita flavorubescens collected from different locations [55].
Apart from the Morąg site, the next pool of B. edulis with elevated level of REEs-when compared to other sites in this study-was from the Augustów Primeval Forest site (id 6) with 758 µg kg −1 dw (Table 1). Interestingly, a similar situation was observed for the mushrooms, M. procera and Cantharellus cibarius (Yellow Chanterelle), from the Augustów Primeval Forest site, which had REE levels well above the median values for these mushroom species collected from other sites across Poland [24]. Notes: ¶ (Sampling site id, see Figure 1); * c, s, w (caps, stipes, whole fruiting bodies, respectively), TLP (Trójmiejski Landscape Park), WLP (Wdzydze Landscape Park), # Number of batches and total number of specimens; § (results for the Warmia-Morąg site "id 8" were not included in calculation of the mean and median values).

LREE and HREE in B. edulis
The light REEs (LREE; here: La, Ce, Pr, and Nd) occurred in mushrooms in higher concentrations when compared to the middle REEs (Sm to Dy) and heavy REEs (from Ho to Lu) ( Table 1). Their median concentrations in the caps, stipes, and whole fruiting bodies followed the same scheme: LREE > MREE > HREE. In detail, the median concentrations (µg kg −1 dw) for these subgroups were 121.4, 12.8, and 5.6 in the caps, 218, 19.1, and 9.2 in the stipes, and 187.7, 17.6, and 8.7 in the whole fruiting bodies. The quotient of LREE/HREE was similar for the morphological parts of B. edulis, i.e., 21.7 for the caps, 23.7 for the stipes, and 21.5 for the whole fruiting bodies. In work by Bau et al., the quotient of LREE/HREE for a single B. edulis specimen (collected in Germany) studied there was 19.6 [25]. Aruguete et al. reported a wide range of concentrations for Nd, La, and Ce between specimens of the same species (Amanita flavorubescens and A. rubescens), with Nd differing from one to three orders of magnitude, and La and Ce up to two orders of magnitude [55].
Hence, the number of individual mushroom specimens collected and tested onsite or contained in a composite sample/pool (sample size) remains essential in characterizing the ability of fungi to bioconcentrate REEs and other minerals and the extent to which they are enriched in the fruiting bodies.  [25,43]. Details of the data, including the REE-normalized patterns are presented in the Supplementary Materials (Figures S1-S4 and Tables S1 and S2). These normalized distributions of REEY in samples of B. edulis showed minor differences in patterns between some of the sites. REE Sc and Y, as non-essential elements, are considered to be co-absorbed by the mycelium alongside Ca and accumulation patterns in mushrooms more or less follow the natural pattern in soil substrata. Thus, some of the detectable differences in the normalized pattern could potentially be reflecting the local soil for a given REE(s).

Shale Normalized Patterns of REEY in B. edulis
For the REEY (Figure 2 and Supplementary Materials)-normalized patterns (caps, stipes, whole B. edulis, and including the literature data), Y showed a small positive deviation in all the plots which in general exhibited an almost flat, unchanging profile. A similar decoupling was observed in studies of mushrooms M. procera and Suillus luteus [22,24]. A strong positive Eu anomaly and abundance of REE were found in B. edulis from the Augustowska Primeval Forest (Supplementary Materials; Table 1) and this can be related to specific nature of the site as observed for M. procera [24]. A positive Eu anomaly was also reported in some vegetation (moss) from south-central Poland [43]. some of the sites. REE Sc and Y, as non-essential elements, are considered to be co-absorbed by the mycelium alongside Ca and accumulation patterns in mushrooms more or less follow the natural pattern in soil substrata. Thus, some of the detectable differences in the normalized pattern could potentially be reflecting the local soil for a given REE(s).  For the REEY (Figure 2 and Supplementary Materials)-normalized patterns (caps, stipes, whole B. edulis, and including the literature data), Y showed a small positive deviation in all the plots which in general exhibited an almost flat, unchanging profile. A similar decoupling was observed in studies of mushrooms M. procera and Suillus luteus [22,24]. A strong positive Eu anomaly and abundance of REE were found in B. edulis from the Augustowska Primeval Forest (Supplementary Materials;

REE Intakes from Bolete Meals
The sum of REEs (∑REE) in the King Bolete varied from 86.7-758 µg kg −1 dw (median 310 µg kg −1 dw; mean ± SD, 327 ± 186 µg kg −1 dw; for conversion to wet weight a divider of 10 can be used assuming moisture content in this species at 90%) for the whole fruiting bodies (Table 1) excluding the very high value of 1796 µg kg −1 dw for the Warmia-Morąg site. Because of the high gourmet value of the King Bolete and the rather unique data on REEs obtained in this study for a relatively large collection of this mushroom, an attempt was made to estimate possible intake of these nonessential elements from theoretical bolete meals. Considering the median ∑REE of 0.031 mg kg −1 wet (whole) weight (0.31 mg kg −1 dw; assuming 90% of moisture) in mushrooms (whole fruiting bodies), a meal of 100 g (whole weight) at one time would expose a consumer to 0.0031 mg of REEs, and consumption of 300 g by high level consumers would result in an intake of 0.0093 mg of REEs. The consumption of such mushroom meals daily for a week would result in the intake of 0.0217 and 0.0651 mg, respectively. Considering the highest ∑REE value of 758 µg kg −1 dw (0.0758 mg kg −1 ww) observed for the Augustów Primeval Forest site (id 6), the consumption of 100 g and a 300 g mushroom meals would respectively result in daily intakes of 0.0076 mg and 0.0227 mg of REEs. Weekly, the consumption of 100 g and 300 g meals of King Bolete collected at Augustów Primeval Forest site would result in intakes of 0.0531 mg and 0.159 mg, respectively.
The effect of culinary processing of mushrooms, including the King Bolete, on the fate of the REEs is unknown. It is anticipated that, as with other elements for which there are data, that depending on a culinary treatment, the concentration in mushroom meals may be smaller, similar, or slightly greater than in the raw unprocessed (wet, whole weight) product. Thus, the assessment is made on the basis of data for the raw (unprocessed) fruiting bodies. The estimated dietary intake (EDI) of REEs from the consumption of King Bolete for an adult of weight 60 kg ranges from 0.014 to 0.126 µg kg −1 day −1 for consumption of 100 g mushroom meal and from 0.043-0.379 µg kg −1 day −1 for consumption of a 300 g mushroom meal. Even for the Warmia-Morąg site, with a highly elevated ∑REE value of 179.6 µg kg −1 ww, the EDI would be 0.299 µg kg −1 day −1 for 100 g and 0.898 µg kg −1 day −1 for consumption of 300 g mushroom (whole weight) meal. These intakes are much lower than the EDI reported in the literature that would be damaging to human health (100-110 µg kg −1 day −1 ) [3,45,46]. Considering the median ∑REE of 31 µg kg −1 ww for whole fruiting bodies, a consumer would need to eat about a 100 g mushroom meal (EDI of 0.0516 µg kg −1 day −1 ) daily for about 1953 days (279 weeks) to exceed the EDI threshold (100 µg kg −1 day −1 ), while for a child this amounts to about 970 days (138 weeks) (EDI of 0.103 µg kg −1 day −1 ).
For a child of weight 30 kg that is able to consume a similar amount as an adult, the EDI ranges from 0.029 to 0.253 µg kg −1 day −1 for consumption of a 100 g mushroom meal and 0.087-0.758 µg kg −1 day −1 for consumption of a 300 g mushroom meal. Moreover, for children consuming boletes from the Warmia-Morąg site, the EDI would be 0.599 µg kg −1 day −1 for a 100 g mushroom meal and 1.796 µg kg −1 day −1 for consumption of a 300 g mushroom meal. Therefore, the health risk assessment of intake of the studied King Bolete shows that the mushroom presents no health risks from REEs, though it would be unusual to see an adult eat as much as a 300 g quantity, even during the peak of the mushroom picking season. Culinary processing of mushrooms (including blanching, boiling, blanching, and pickling) lowers the amount of the metallic elements in mushroom meals (wet weight basis) [56][57][58], while frying or braising to some degree prevents loss or slightly enhances their quantity on a wet (whole) weight basis, as has been observed in the case of Hg, 137 Cs, and 40 K [59][60][61]. Even after consumption, intestinal absorption of metallic elements from a mushroom (culinary processed or only dried) is generally considered to be below 50 % [58].

Conclusions
The literature on REE in foodstuffs, and mushrooms in particular, is scarce. This study documented the rare-earth element contents of the popular and valued edible mushroom, the King Bolete (B. edulis), foraged across Poland. Results of this study show rather low contents of REEs in the whole mushroom and in the separate morphological parts (caps and stipes). Available data also show that the possibility of contamination of the fruiting bodies of B. edulis by rare-earth elements from their increased usage worldwide in the last decades seems unlikely. Thus, from the point of view of REE contamination, intake of this mushroom, even for high level consumers, would not present any known toxicological risks.
Supplementary Materials: The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijerph19158948/s1, Table S1: The NASC-, PAAS-, EUS-and WSH-normalized REE concentrations in the caps, stipes and whole fruiting bodies of B. edulis (whole set). Table S2: NASC-, PAAS-, EUS-and WSH-normalized REE concentrations in the whole fruiting bodies of B. edulis from the Morąg and Augustów sites in this study and a single specimen of B. edulis examined by Bau et al. (2018). Figure