Accumulation and distribution of ⁴⁰K in the chaga mushroom

Abstract

This work is the first report on activity concentrations of ⁴⁰K in Inonotus obliquus sampled in a virgin forest of Siberia. The results have shown that the chaga conk is characterized by high activity concentrations of ⁴⁰K, averaging 1,641 Bq/kg dry weight (DW) and peaking at 3,502 Bq/kg DW. Activity concentrations of ⁴⁰K in chaga conks have been defined to increase from the near-trunk stratum to the crust of the conk with increased exposure to the solar radiation. Our measurements have revealed the samples to be mildly contaminated with ¹³⁷Cs. Intensive assimilation of ⁴⁰K by chaga conks has been shown as a normal and innate feature of the wild chaga mushroom.

The chaga mushroom Inonotus obliquus, mainly associated with boreal forests, is one of a few natural substances, recognized by traditional medicine as a chemopreventive remedy and appreciated for its other healing properties (Desai et al., 2008; Wang et al., 2012). I. obliquus is a long-living and wood-destroying parasitic fungus hosted predominantly by species of the birch family Betulaceae. Its mycelium propagates into the trunk, occupies the entire duramen, and the bulk of the radial wood. Having reached the surface of the trunk, the mycelium starts to produce an outeral sterile conk, slowly growing over the years. The conk is referred to as "chaga", once it has been harvested, distributed and used in medicine (other less common names are the clinker polypore, cinder conk, birch mushroom, and black birch touchwood).

Being widely distributed in boreal forest areas, I. obliquus is one of the most popular items used by traditional medicine in Northern Eurasia. Without regard to its medicinal nature, the chaga has long been recognized by tisane drinkers. Chaga is collected and processed into various types of products, and its derivatives are widely promoted and marketed. In Russia, chaga is available as syrups, creams, extracts, tisanes, and tinctures in pharmacies, and it is also offered as raw materials. Chaga is favored and used in food and medicine by local residents in the Asia-Pacific region, notably, in China and South Korea. The demand for chaga is stimulated by numerous biochemical and pharmacological studies, that have made a considerable contribution into understanding of curative properties of the conk. Moreover, recent works on chaga have revealed antioxidative (Hu, Zhang, Lei, Yang, & Sugiura, 2009; Liang, Zhang, & Wang, 2009), antiproliferative and antimutagenic (Ham et al., 2009), anticancer (Nomura, Takahashi, Uesugi, Tanaka, & Kobayashi, 2008; Youn et al., 2008; Kuriyama et al., 2013; Lee, Lee, Kim, Song, & Hong, 2014; Kang, Jang, Mishra, & Lee, 2015), antitumor (Song et al., 2008), anti-inflammatory (Sung et al., 2008), antiviral (Shibnev et al., 2011), antimicrobial (Harikrishnan, Balasundaram, & Heo, 2012; Glamočlija et al., 2015), antidiabetic (Wang et al., 2017), anti-anaphylactic (Yoon et al., 2013), and immunomodulatory (Chen, Huang, Cui, & Liu, 2015; Glamočlija et al., 2015) properties of chaga and its organic compounds.

Radioactivity of chaga, one of the most significant features of such natural substances, requires standardization. While organic compounds and pharmacological activity of chaga extracts and isolated components of chaga are subject to extensive examination, the ultimate isotopic composition of chaga, that determines both its beneficial, and injurious properties, is addressed less comprehensively (Shivrina, Lovyagina, & Platonova, 1959; Popov & Shpan'ko, 2000; Lee et al., 2008). As distinct from many mushroom species studied with respect to their radioisotope content (Kalac, 2001; Turhan, Köse, & Varinlioğlu, 2007; Mietelski, Dubchak, Błażej, Anielska, & Turnau, 2010; Gwynn, Nalbandyan, & Rudolfsen, 2013; Yamada, 2013; Trappe, Minc, Kittredge, & Pink, 2014; Falandysz et al., 2015; Ohnuki et al., 2016; Falandysz, Zhang, & Zalewska, 2017), chaga radioactivity appears to have been assessed only in one scientific paper. Regrettably, the work gives no details of sampling locations, denoted merely as "Russia", "China", and "N. Korea", as the samples have been obtained from open markets (Lee et al., 2008). A growing demand for chaga products and expansion of the chaga market spur efforts to identify potential chaga resources and to cultivate the fungus (Vanhanen, Peltola, Ahtikoski, & Pappinen, 2014; Ka, Jeon, Park, Lee, & Ryu, 2017). Today, chaga grown under extremely heterogeneous conditions enters into the market. Our study is aimed at providing information regarding the natural radioactivity of chaga collected in Eastern Siberia, based on total potassium accumulation by the conk (Shivrina et al., 1959; Popov & Shpan'ko, 2000). We have also measured ¹³⁷Cs in chaga to evaluate anthropogenic impacts on the study area.

The study areas are located in the eastern part of the Lena river basin, Eastern Siberia, Russia. This is an underpopulated region characterized by sparse, small-scale settlements, mining industry, public works infrastructure, and spacious wildland areas (Degtyarev, 2016). Due to its geographical location, climate, and significant orographic isolation, the region is least exposed to global fallout. The region is located beyond the impacts of peaceful underground nuclear explosions performed in Yakutia (Chevychelov & Sobakin, 2017; Goryachenkova et al., 2017).

White-barked tree species of the birch of the Lena basin are represented by Betula pendula, B. pubescens, and B. ermannii. Betula pendula, silver birch, is ubiquitous and is the most well-known host of I. obliquus. Betula pendula occurs, as a rule, in mixed forests in taiga, in post-fire and post-logging communities, and, rarely, as birch groves. Birch groves, their size ranging from a few square meters to several hectares, are patchily distributed over wide river valleys. They form parts of wetlands usually surrounding lakes, marshes, and bogs.

We have collected 57 chaga conks from 11 locations, 1–300 km apart, in the Central Yakutian plain (62°14'N, 129°5'E; сlayic turbic cryosols) and the Yudomo-Maiskoe upland (59°24'–60°19'N, 133°4'–135°3'E; сlayic, and fluvic turbic cryosols) in 2012–2018. Our five samples have been collected in upland woodlands (сlayic turbic cryosol, mixed forests) on 25–40 y old birch trees, and the rest in riparian woodlands (fluvic turbic cryosol, mixed forests, and birch groves) on 25–70 y old birch trees. Five conks have been found growing on one trunk. Three dead conks have been found on three dead birch trees. We have also collected a chaga basidiocarp, unaffected wood, bark with phloem from four cross-sections of the affected birch logs, 18 samples taken from uninfected 10–60 y old birch trees, and 18 fruiting bodies of Fomes fomentarius, a polypore and saprophyte, from dead birch trees. The shading and aspect of the collected conks have been identified in the process of sampling. Here, the term "aspect" refers to the compass direction that a conk faces. We determine a conk aspect as the position of the conk center as an angle measure, clockwise and counterclockwise to 180° from a southerly direction, as the reference point (0°). The scale interval is 5°. Prior to being dried, five larger conks have been prepared for a layer-by-layer analysis by their separation into the crust (less than 1 cm-size hardened and crumbly outer dead tissue), the middle stratum (3–5 cm-size), and the innermost near-trunk stratum (2–3 cm-size). One affected birch tree has been dissected, and the volume of the chaga basidiocarp has been measured. The density of dried tissue of the basidiocarp has been detected. Based on the obtained values of the volume and density, the dry weight of the basidiocarp has been calculated. The samples have been oven-dried to the constant weight, grinded and analyzed by the scintillation gamma spectrometer (Scientific Production Company "Doza", Ltd, Zelenograd, Russia), using the NaI(Tl) Ø63 × 63 mm detector (the minimum detectable activity 40 Bq for ⁴⁰K and 3 Bq for ¹³⁷Cs; the reference standard IEC 61563; the uncertainty of determination ±10%). The value below the minimum detectable activities has been taken as zero.

The maximum and average activity concentrations of ⁴⁰K in the collected conks are much higher, as compared with those in the basidiocarp of F. fomentarius (Table 1), and are comparable with those in many other fungi in Eurasia. Some of them, e.g., Hygrophoropsis aurantiaca and Tricholoma terreum, are capable of concentrating up to 4,000 Bq/kg dry weight (DW) (Kalac, 2001; Turhan et al., 2007; Mietelski et al., 2010; Falandysz et al., 2015, 2017). As the activity concentrations of ⁴⁰K of the three dead conks from dead birch trees fall within the range observed in living conks, these concentrations are maintained for a long time after the tree and the conk have died (Table 1).

Table 1. Measured activity concentrations of ⁴⁰K in the samples (Bq/kg DW).

Sample	Sample size	Mean value	Standard error	Minimum value	Maximum value	Value range	Median value	Standard deviation
Chaga conk	52	1,641	103	509	3,502	2,993	1,500	741
Chaga conk sampled in the Central Yakutian plain	4	1,636	230	1,326	2,316	990	1,450	459
Chaga conk sampled in the Yudomo-Maiskoe upland	48	1,642	110	509	3,502	2,993	1,509	763
Chaga conk sampled in riparian woodlands	47	1,650	112	509	3,502	2,993	1,519	769
Chaga conk sampled in upland forests	5	1,563	192	1,276	2,316	1,040	1,400	429
Chaga conk grown on Betula pendula	51	1,651	114	509	3,502	2,993	1,500	745
Chaga conk grown on Alnus hirsute	1	1,149	–	–	–	–	–	–
Chaga conk grown on birch in sparse wood edge	5	2,781	269	2,023	3,502	1,479	2,625	601
Chaga conk grown on birch shadowed by conifers	4	1,185	256	509	1,693	1,184	1,269	512
Dead chaga conk collected from dead birch	3	1,662	812	670	3,272	2,602	1,044	1,407
Chaga conk crust	5	2,684	3,96	1,248	3,548	2,300	2,943	885
Chaga conk middle stratum	5	1,756	405	925	1,866	2,305	1,631	906
Chaga conk near-trunk stratum	5	1,045	138	703	1,491	788	961	309
Chaga conk in cross-section of affected birch	4	1,555	212	1,158	2,143	985	1,459	423
Chaga basidiocarp in cross-section of affected birch	4	356	126	0	642	642	318	281
Radial wood in cross-section of affected birch	4	46	46	0	187	187	0	94
Bark with phloem in cross-section of affected birch	4	127	89	0	475	475	72	199
Wood of non-infected birch	18	23	12	0	159	159	0	49
Fomes fomentarius basidiocarp	18	31	19	0	324	324	0	79

The obtained data have revealed that activity concentrations of ⁴⁰K in the conk depend on the aspect and shading of the conk. The ⁴⁰K values highly correlate (r = − 0.62) with the angle of the conk position in relation to the southerly direction, and they decrease with the conk displacement from the southerly to the northerly aspect (Figs. 1, 2). This is also apparent from the variety of radionuclide activity concentrations in five conks growing on the same trunk (Fig. 3). Also, higher values of ⁴⁰K have been observed in conks grown on well-lighted (sparse or isolated) trees on the edge of birch forests, and, vice versa, its lower values have been found in conks from mixed forests, where infected birches are shaded by conifers (Table 1). We assume that ⁴⁰K accumulation increases with an increased exposure to sunlight. Higher insolation heats the conk, thus intensifying evaporation and sap inflow. The morphology of the conk itself suggests intense water metabolism. Due to continuous weathering and cracking, the crust of the chaga conk is dull-black and crumbly, thus developing higher sunlight-absorption and the larger evaporation surface.

Fig. 1. Variations of activity concentrations of ⁴⁰K in reference to aspect changes of conk in the chaga.

Fig. 2. Variations of activity concentrations of ¹³⁷Cs in reference to aspect changes of conk in the chaga.

Fig. 3. Variations of activity concentrations in reference to aspect changes of conks grown on one trunk in the chaga.

A layer-by-layer analysis has revealed activity concentrations of ⁴⁰K in the samples of chaga conks to be unevenly distributed. ⁴⁰K values here strongly correlate (r = 1.00) with the layer position, and increase from the near-trunk stratum to the crust (Table 1). The crust is dead tissue consolidated by weathering, and the highest levels of ⁴⁰K activity concentration in the crust result from external physical impacts that have densified the crust (the density of the inner tissues and the crust of the conk are 0.2–0.3 g DW/cm³ and 1.0–1.1 g DW/cm³, respectively).

The analysis of the wood of the non-infected birch trees and a cross-section of the birch log affected by I. obliquus has shown that the fungus accumulates ⁴⁰K in its basidiocarp. The intensive assimilation of ⁴⁰K has been found to be in agreement with intensive assimilation of the total potassium (up to 10% of ash has been measured) by the conk (Shivrina et al., 1959; Popov & Shpan'ko, 2000), which is a normal physiological process for the fungus. Extremely low activity concentrations of ⁴⁰K in the wood of the non-infected birch trees and the cross-sections of the affected birch logs have shown that I. obliquus absorbs ⁴⁰K mainly from the sap passing through tree tissues (Table 1). This is readily illustrated by the ratio of activities in the basidiocarp and the wood utilized by the fungus. The dissected birch affected by I. obliquus has the conk of 100 g (140 Bq) and contains the 11,000 cm³ basidiocarp. Judging by the measured density (0.36 g DW/cm³), the weight of the basidiocarp makes 4,000 g (about 2,600 Bq). The unaffected wood of the dissected birch of the same weight reveals the activity below 120 Bq. Thus, having digested the volume of the host wood equal to the aggregated volume of the basidiocarp and the conk, the chaga mushroom can not have obtained 2,740 Bq (140 Bq + 2,600 Bq). This implies that the true transfer factor should be calculated in relation to the tree sap.

Unlike ⁴⁰K, the activity concentrations of ¹³⁷Cs in all our samples are low (Table 2), thus substantiating the inferences that global fallout has had minor impacts on ecosystems of the region (Chevychelov & Sobakin, 2017). The mean value of activity concentrations of ¹³⁷Cs in the conk agrees with the lowest values measured in chaga obtained from Russian and Chinese markets (11–13 Bq/kg DW), and ranks slightly above the activity concentrations in North Korean samples (3 Bq/kg DW) (Lee et al., 2008). It should be noted that chaga conks without the crust may be available on the market, i.e., a conk containing the maximal activity concentration of ¹³⁷Cs may have been peeled off from its crust. The mean value of ¹³⁷Cs in the collected chaga is equal to the lowest values or incomparably low values reported for short-living fungi (Korky & Kovalski, 1989; Bakken & Olsen, 1990; Kalac, 2001; Lee et al., 2008; Turhan et al., 2007; Mietelski et al., 2010; Gwynn et al., 2013; Yamada 2013; Trappe et al., 2014; Falandysz et al., 2015; Ohnuki et al., 2016). The activity concentrations of ¹³⁷Cs in the chaga conks, like those of ⁴⁰K, strongly correlate (r = 0.95) with the layer position, revealing minimal values in the near-trunk stratum and maximum in the crust (the conk outer layer). ¹³⁷Cs, as an artificial radionuclide, occurs in low quantities in areas least exposed to global fallout. ¹³⁷Cs, being a contaminant, is distributed unevenly in natural ecosystems, and the values of its activity concentrations in chaga show less evident correlations (r = 0.14) with the conk aspect and shading (Table 2; Figs. 2, 3). While they are chemical analogues, ¹³⁷Cs and ⁴⁰K are supposed to differ in biological metabolism, as these isotopes reveal different patterns of transfer and accumulation in the soil–plant system (Marčiulioniene, Lukšiene, & Jefanova, 2015). The similar conclusion has been drawn from a study on mushrooms. A lack of correlations between ¹³⁷Cs and ⁴⁰K suggests different uptake mechanisms for two elements. Potassium, including ⁴⁰K, is self-regulated by physiological requirements of mushrooms. The radiocaesium content largely depends on the substratum from which the mushrooms take nutrients (Guillén & Baeza, 2014; Kalac, 2019).

Table 2. Measured activity concentrations of ¹³⁷Cs in the samples (Bq/kg DW).

Sample	Sample size	Mean value	Standard error	Minimum value	Maximum value	Value range	Median value	Standard deviation
Chaga conk	52	10.5	1.8	0	64.2	64.2	7.2	12.7
Chaga conk sampled in the Central Yakutian plain	4	7.3	4.3	0	15.8	15.8	6.8	8.5
Chaga conk sampled in the Yudomo-Maiskoe upland	48	10.7	1.9	0	64.2	64.2	7.2	13.0
Chaga conk sampled in riparian woodlands	47	10.6	1.9	0	64.2	64.2	6.9	13.1
Chaga conk sampled in upland forests	5	9.1	3.7	0	16.2	16.2	13.5	8.4
Chaga conk grown on Betula pendula	51	10.5	1.8	0	64.2	64.2	6.9	12.8
Chaga conk grown on Alnus hirsute	1	9.2	–	–	–	–	–	–
Chaga conk grown on birch in sparse wood edge	5	10.2	4.5	0	22.1	22.1	8.1	10.0
Chaga conk grown on birch shadowed by conifers	4	17.9	7.1	0	34.7	34.7	18.4	14.2
Dead chaga conk collected from dead birch	3	13.1	6.7	0	22.1	22.1	17.2	11.6
Chaga conk crust	5	10.4	2.6	3.0	18.4	15.4	11.8	5.9
Chaga conk middle stratum	5	9.4	5.1	0	28.6	28.6	7.2	11.3
Chaga conk near-trunk stratum	5	6.0	2.2	1.9	13.5	11.6	4.1	4.9
Chaga conk in cross-section of affected birch	4	0	–	–	–	–	–	–
Chaga basidiocarp in cross-section of affected birch	4	10.6	3.6	0	19.9	19.9	12.5	8.0
Radial wood in cross-section of affected birch	4	3.7	2.6	0	11.0	11.0	1.8	5.2
Bark with phloem in cross-section of affected birch	4	12.4	4.8	4.0	30.5	26.5	8.7	10.8
Wood of non-infected birch	18	3.5	1.2	0	17.3	17.3	1.6	4.9
Fomes fomentarius basidiocarp	18	12.1	8.9	0	148.0	148.0	0	37.7

High values of ⁴⁰K in chaga appear worthy of investigation in view of its curative properties, notably, the possible contribution of certain levels of ⁴⁰K activity concentrations, along with organic substances, to chaga antitumor and other medicinal properties and the possible side effects in administration of chaga as medicine. We have found that accumulations of ⁴⁰K and ¹³⁷Cs in the chaga conk depend on the conk aspect, and increase with its exposure to sunlight. The activity concentrations of ⁴⁰K in the conk have been identified to be unevenly distributed and to increase from the near-trunk stratum to the crust. The results provide a good indication of suitable materials for selection of raw chaga, a way to obtain chaga tissues with various activity concentrations of ⁴⁰K, and a basis for setting requirements and elaborating guidelines for chaga collecting.