210Po and 210Pb in King Bolete (Boletus edulis) and Related Mushroom Species: Estimated Effective Radiation Dose and Geospatial Distribution in Central and Eastern Europe

210Po and 210Pb occur naturally and are the most radiotoxic isotopes of the uranium (U) decay chain. Samples of Boletus edulis and related mushroom species, including B. pinophilus, B. reticulatus, B. luridus and B. impolitus, collected from Poland and Belarus were investigated for the activity concentrations of these isotopes and also for their potential health risk through adult human consumption. The results showed that spatially, the occurrence of 210Po and 210Po was heterogeneous, with activities varying from 0.91 to 4.47 Bq∙kg−1 dry biomass and from 0.82 to 5.82 Bq∙kg−1 db, respectively. Caps and stipes of the fruiting bodies showed similar levels of contamination. Consumption of boletes foraged in Poland could result in exposure to a combined radiation dose of 10 µSv∙kg−1 db from both isotopes. This dose is not significant compared to the total annual effective radiation dose of 210Po and 210Pb (54–471 µSv∙kg−1) from all sources, suggesting that these mushrooms are comparatively safe for human consumption.


Introduction
Dried mushrooms (carpophores of Basidiomycota) are relatively rich in mineral constituents (the water content of fresh mushrooms is around 90%), but radioactive elements can form a significant part of these and pose a health risk to consumers. A key example is the contamination of wild mushrooms with artificial nuclides of radiocaesium ( 134/137 Cs) after nuclear cataclysms. These were the most commonly studied nuclides in mushrooms as documented and reviewed [1][2][3][4][5][6][7][8][9]. 210 Po and its parent nuclide 210 Pb originate from the 238 U (uranium) decay chain. They are also the most toxic amongst the uranium chain radioactive elements, and their half-lives are 138.38 days for 210 Po and 22.3 years for 210 Pb [10][11][12]. 210 Po, 210 Pb, 226 Ra and 40 K as natural nuclides contribute mainly to the effective radiation background in biota that are unexposed to anthropogenic radioactivity: 210 Po + 210 Pb + 226 Ra annually contributes 165 µSv to a daily diet, while 40 K provides 140 µSv [13].
The family of the Boletus fungi is rich in genera and species that are cosmopolitan and collected worldwide [14,15]. The majority from the genus Boletus are edible (only a few are toxic, e.g., Rubroboletus satanas), tasty, and valued by local consumers. Perhaps the most remarkable and prized of these, the King Bolete, Boletus edulis, occurs relatively frequently and is native to forests of the temperate climate [14].
The objectives of this study were to evaluate the occurrence, distribution within fruiting bodies and possible risk (to consumers) from 210 Po and 210 Pb that tend to accumulate in Boletus edulis, Boletus pinophilus, Boletus reticulatus, Boletus luridus and Boletus impolitus mushrooms and also to prepare, based on the results, interpolation maps to spatially characterize the occurrence of both nuclides in boletes in Poland.

Materials and Methods
The bolete mushrooms studied, species such as B. edulis, B. pinophilus, B. reticulatus, B. luridus and B. impolitus, were collected from 25 woodlands/forested sites across Poland (the Pomerania, Kujawy, Warmia, Podlasie and Masuria regions, and the Tatra and Sudety Mts.). Also included in the study were B. reticulatus samples from two locations in Belarus (Gomel and Minsk regions) from our depository. To obtain an insight into the 210 Po and 210 Pb distribution within the fruitbody, some mushroom samples were separated into cap and stipe during preparation (samples/pools id. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17]. For these, the results of 210 Po and 210 Pb activity concentrations in the whole specimens were calculated based on the activity concentration and biomass (caps and stipes) percentage share in the fruiting bodies (Table 1). Each analytical sample of boletes (4-5 g) had been spiked with 10 mBq of 209 Po before radiochemical analysis as an internal tracer, and all prepared samples were digested using a concentrated solution (65%) of nitric acid (HNO 3 ) [16]. The residues obtained were dissolved in 0.5M solution of HCl with added ascorbic acid. The activity concentration of 210 Pb in analyzed samples was calculated indirectly via the activity measurement of its daughter 210 Po. After at least six months of deposition time, the activities of the ingrown 210 Po was measured in an alpha spectrometer (Canberra-Packard, USA). 210 Pb activities measured in the studied boletes were calculated at their time of collection using the simplified equation for the daughter activity as a function of time [17]. The 210 Po and 210 Pb yield in the analyzed mushroom and soil samples ranged from 90 to 98%. The measurement results of 210 Po and 210 Pb activity concentrations were given with standard deviation (SD) calculated for 95% confidence intervals. The method's accuracy was assessed using an IAEA reference material (IAEA-414) and participation in IAEA intercomparison exercises were estimated at better than 95%. Because of the abnormal distribution of radionuclides, non-parametric tests were used (U-test Mann-Whitney and H-test Kruskal-Wallis) to assess the significance of results, and the most important level achieved was quoted. Generally, the defined significance level was p = 0.05. The interpolation maps were prepared using QGIS software (QGIS Development Team) and results for the whole mushrooms.
An important aspect of chemical contaminants in biota is their uptake and distribution in the species. It has to be mentioned that the vegetative (main) body of basidiomycetes is the mycelium that is buried in the substrate, while the fruiting body (the mushroom) is an ephemeral reproductive organ used for dispersing spores into the surrounding space. Since the collection of a mycelium under natural forest conditions is generally discouraged by local customs (and regulation in some cases) because of the apparent potential damage to the habitat, it was not included in our study. Thus, to know approximately the distribution/localization of an element in a mushroom (sometimes the only cap is suitable or used for the culinary purpose) and to calculate its bioconcentration factor, a mushroom is separated into cap and stipe, which are examined individually. Therefore, it is possible to calculate, in the simplest mathematical way (no presentation of an equation/formula is necessary), the quotient of the occurrence (distribution) of an element within a specimen, expressed as the Q C/S index (cap to stipe using normalized results for fully dehydrated materials).
The value of Q C/S index > 1 (sometimes also called as distribution ratio, DR) shows that an element is preferentially accumulated in the caps [2][3][4][5][6][7]11,18,19]. The pattern of 210 Po and 210 Pb allocation in the morphological parts may change while ageing (maturing) [20], although mushrooms are generally not consumed when they reach this stage.

210 Po and 210 Pb Activity Concentrations in Boletes
The activity concentrations determined in the bolete samples from Poland and Belarus showed a heterogeneous distribution of 210 Po and 210 Pb ( Table 1). The activity concentrations (of 210 Po and 210 Pb, respectively) in whole mushrooms were in the range from 0.91 ± 0.10 Bq·kg −1 db in Wysokie, to 4.47 ± 0.28 Bq·kg −1 db in Osowa, and from 0.82 ± 0.09 Bq·kg −1 db (Wysokie) to 5.82 ± 0.32 Bq·kg −1 db (Elbląg), respectively. The results of 210 Po and 210 Pb activities in the Belarusian samples were similar to those in Poland (Table 1).
These activities (  [12,[21][22][23][24][25][26][27]. The statistical analysis showed a lack of significant differences in 210 Po or 210 Pb activity concentrations among the five bolete species (H-test Kruskal-Wallis p-value 0.33 for 210 Po and 0.37 for 210 Pb). There was also a lack of significant differences in the distribution of the nuclides between caps and stipes for each individual species and sampling point (U-test Mann-Whitney p-value 0.85 for 210 Po and 0.59 for 210 Pb). The bioconcentration in terrestrial species depends on the geochemical background and atmospheric fallout, but data for these parameters were not available for the boletus samples in the present study. The activities of 210 Po and 210 Pb in examined B. edulis, B. pinophilus, B. reticulatus, B. luridus and B. impolitus can be considered as low, suggesting that these species, similar to mushrooms of other genera from the Boletaceae family examined previously, namely the genus Leccinum and Leccinellum, do not strongly bioconcentrate 210 Po and 210 Pb [11,18,19]. However, again, this will depend to some extent on the background. Previously, Gwynn et al. have reported that differences in 210 Po activity concentrations for individual specimens of the same mushroom species from the same stand were generally less than a factor of 3 in most cases [22].
In an attempt to visualize the spatial occurrence of 210 Po and 210 Pb activities in five bolete species in Poland, the activities were mapped in Figures 1 and 2. 210 Po mushrooms from the northern and north-eastern regions of the country (Figure 1) appeared to be more contaminated, while the occurrence of 210 Pb was more heterogeneous (Figure 2). Samples collected from northern and north-eastern regions were relatively more contaminated. The 210 Pb spatial occurrence was similar to that noticed earlier for the Parasol Mushroom Macrolepiota procera [28], although activity concentrations were lower in boletes. This may be explained by a number of factors, such as the natural elemental occurrence in the soil bedrock, the depth of mycelium penetration in the substrate and also by the feeding behavior and/or nuclide enrichment in the organic matter of the soil (Macrolepiota are saprophytic and favor habitats that are rich in decaying organic matter). The boletes are mutual symbionts associated with a specific plant's rhizosphere root system, and their interactions with trees, soil substrate and soil solution are more complicated [29]. As mentioned, the levels and distribution of both 210 Pb and 210 Po activity concentrations in the upper soil layers can be associated not only with the parent bedrock, but to some degree also with atmospheric fallout, where they are the result of the precipitation of radon decay products from the atmosphere and the level of 210 Pb and 210 Po contained in the top layer of soil can be correlated with the amount of atmospheric precipitation [10,30].

Distribution of 210 Po and 210 Pb within a Fruitbody
Analysis of the distribution of 210 Po and 210 Pb within the fruiting bodies of the boletes showed a wide range of Qc/s values, i.e., 0.60-1.67 for 210 Po (mean value 1.00 ± 0.19 and median 1.07 ± 0.16) and 0.55-1.52 for 210 Pb (mean 0.93 ± 0.18 and median 0.93 ± 0.13). There were no statistically significant differences between concentrations in the caps and stipes (U-test Mann-Whitney p-value 0.76) ( Table 2). Generally, 210 Po is more mobile in soil than 210 Pb and easier bioconcentrated in fruiting bodies by some Basidiomycota [12]. Lead, including 210 Pb, is known to be weakly bioconcentrated by numerous Basidiomycota studied so far, while stable lead is a notorious soil pollutant because of the legacy of historical industrial pollution and also current emissions from metal (lead, copper, zinc) smelters and other use [31][32][33][34][35][36]. Thus, sometimes, a relatively elevated concentration of stable Pb observed in mushrooms is due to the high degree of substrate soil pollution [31,32,34]. This does not apply to 210 Pb that typically occurs at low activity concentration levels in mushrooms, as seen in this and other studies [27,[35][36][37].

Annual Effective Radiation Doses for Adults
Based on the calculated 210 Po and 210 Pb content in dried boletes, the effective radiation doses were estimated (in 10 kg of an equivalent fresh mushroom portion) to identify their potential radiotoxicity to consumers (Table 3).

Distribution of 210 Po and 210 Pb within a Fruitbody
Analysis of the distribution of 210 Po and 210 Pb within the fruiting bodies of the boletes showed a wide range of Qc/s values, i.e., 0.60-1.67 for 210 Po (mean value 1.00 ± 0.19 and

Distribution of 210 Po and 210 Pb within a Fruitbody
Analysis of the distribution of 210 Po and 210 Pb within the fruiting bodies of the boletes showed a wide range of Qc/s values, i.e., 0.60-1.67 for 210 Po (mean value 1.00 ± 0.19 and   For adults, the effective dose conversion coefficients (dose per unit exposure) for 210 Po and 210 Pb ingestion that ICRP recommends for the calculation of equivalent and effective doses are 1.2 and 0.69 µSv·Bq −1 , respectively [38]. In the case of the bolete samples in this study, the consumption of whole mushrooms could lead to an effective 210 Po radiation dose of 1.09 ± 0.12 to 5.37 ± 0.34 µSv·kg −1 db with a corresponding dose of 0.57 ± 0.06 to 4.02 ± 0.22 µSv·kg −1 db from 210 Pb decay.

Conclusions
This study demonstrates that B. edulis, B. pinophilus, B. reticulatus, B. luridus and B. impolitus accumulate 210 Po and 210 Pb at different concentrations. The interpolation maps suggest a non-uniform spatial distribution of these nuclides based on their occurrence in common edible mushrooms. The occurrence indicates the geographical distribution of these nuclides across Poland, which also shows noticeable agreement with the natural radiological background. Morphologically, the 210 Po and 210 Pb quotients between cap and stipe (Q c/s ) ranged from 0.55 to 1.67. Consumption of the analyzed mushrooms would result in a dose of 10 µSv·kg −1 db in total, from both 210 Po and 210 Pb, which would not contribute significantly to the total annual effective radiation doses from 210 Po and 210 Pb intake from other sources for adult consumers. This suggests that consumption of these mushrooms is comparatively safe from the radiological protection point of view.  Institutional Review Board Statement: Not applicable-this article does not contain any studies with human participants or animals.