There is no scientific consensus regarding the safety of the Yellow Knight mushroom Tricholoma equestre (L.) P.Kumm. Following reports of cases of intoxication involving effects such as rhabdomyolysis, and supportive observations from in vivo experimental models, T. equestre is considered as a poisonous mushroom in some countries while in others it is still widely collected from the wild and consumed every year. In this paper, we review all the available information on T. equestre including its morphological and molecular characterization, nutritional value, levels of contaminants observed in fruiting bodies, the possibility of mistake with species that are morphologically similar, and the in vivo data on safety and cases of human intoxication. Based on available data, it is suggested that T. equestre cannot be considered as a toxic species and does not appear to exhibit any greater health threat than other mushroom species currently considered as edible. More care should be taken when reporting cases of human poisoning to fully identify T. equestre as the causative agent and to exclude a number of interfering factors. Specific guidelines for reporting future cases of poisoning with T. equestre are outlined in this paper. Any future research involving T. equestre should present the results of molecular phylogenetic analyses.

Introduction Mushrooms are an important food product valued for their taste, delicacy, nutritional value, and biological activity, which is currently being extensively researched (Aly, Debbab, & Proksch, 2011; Rathore, Prasad, & Satyawati, 2017; Reis, Martins, Vasconcelos, Morales, & Ferreira, 2017). Although there is a growing interest in cultivated forms, the collection of wild mushrooms has a long tradition in various regions of Europe (particularly in Slavic countries), Asia, and North America, and is still practiced by many individuals (Mortimer et al., 2012; Peintner et al., 2013). Collecting wild mushrooms for consumption is, however, associated with a risk of poisoning arising from the ingestion of toxic species, often of similar morphological appearance to those considered as edible. Depending on toxin, its dose and individual susceptibility or associated conditions (for example, simultaneous consumption of alcohol), the clinical symptoms can widely vary in onset time and the magnitude of their manifestation, encompassing mild or severe gastrointestinal irritation, vomiting, headache, fatigue, hallucinations, seizures, hemolysis, and life‐threatening liver or renal damage (Chen, Zhang, & Zhang, 2014; Graeme et al., 2014). Every year, ingestion of toxic mushrooms causes various health disturbances, and some can lead to death in the absence or in spite of medical intervention. The most poisonous species include those producing amatoxin peptides (with α‐amanitin revealing the greatest toxicity) such as Amanita phalloides (Vaill. ex Fr.) Link, A. virosa (Fr.) Bertillon and A. bisporigera G.F. Atk., Galerina marginata (Batsch) Kühner and Conocybe filaris (Peck) Singer which cause approximately 50 deaths annually in Europe and Asia (Pilz & Molina, 2002; Vetter, 1998). Since medieval times, Tricholoma equestre (syn. T. flavovirens, (Peerson), and syn. T. auratum (Paulet) Gillet) commonly known as the Yellow Knight mushroom or Man on Horseback, has been widely considered as an edible species in various geographical locations, with no scientific or anecdotal evidence of any potentially toxic effects. This view, however, was suddenly undermined in 2001 when Bedry et al. published a highly publicized paper in the New England Journal of Medicine entitled “Wild‐mushroom intoxication as a cause of rhabdomyolysis.” This brief report described a total of 12 clinically relevant cases that occurred in France and involved intoxication with T. equestre, some with a fatal outcome. The major clinical effect observed in the poisoned individuals included muscle injury biochemically marked by significantly increased levels of serum creatine kinase. These observations were also supported by in vivo rodent experiments involving 3‐day exposure to powdered or extracted fruiting bodies of T. equestre that reported an increase in serum creatine kinase and disorganization of muscle fibers. The work of Bedry et al., 2001 was later followed by series of case reports of T. equestre poisoning from Poland (Anand & Chwaluk, 2010) and Lithuania (Laubner & Mikulevičienė, 2016), as well as data from in vivo toxicological assessment (Nieminen, Kärjä, & Mustonen, 2008; Nieminen, Mustonen, & Kirsi, 2005). Considering the available and growing evidence of the toxicity of T. equestre, a number of countries have officially registered T. equestre as a poisonous species (Bedry & Gromb, 2009) (Figure 1). Nevertheless, it is still considered as an edible mushroom in some parts of Asia, Europe, and North America, although a number of locally published amateur mushroom guidelines contain a warning that this species can cause clinical poisoning. Being ectomycorrhizal, T. equestre is not commercially cultivated but in Europe, particularly in its central part, fruiting bodies collected from the wild are seasonally sold on the market (Kasper‐Pakosz, Pietras, & Łuczaj, 2016). Figure 1 Open in figure viewer PowerPoint The status of edibility of Tricholoma equestre in European countries. Red indicates countries regarding it as inedible/poisonous species, green indicates countries considering it as edible/conditionally edible. Data based on national legislations (if enforced) or information from local mycologists. The lack of a consensus on the safety of T. equestre creates an urgent need to comprehensively evaluate available evidence, yet such an assessment is missing. Therefore, in the present review we summarize information on the morphological and molecular features of this mushroom species, its distribution, habitat, nutritional value and reported levels of contaminants, discuss the available clinical and experimental data on its toxicity, present a critical viewpoint questioning the concerns over its edibility, propose some guidelines to be followed when reporting any future cases of intoxication with this mushroom species, and highlight future prospects in the field of T. equestre research.

Distribution and Habitat T. equestre shows a wide distribution encompassing Europe, North America, Central Asia, and Japan. It is a mycorrhizal mushroom associated particularly with coniferous tree species (mainly Pinus rarely Abies and Picea), and often associated with nutrient‐ and humus‐poor sandy soils. Similarly to many other representatives of the Tricholoma genus, it prefers cooler conditions and occurs in the highest frequencies in northern forests and higher altitude habitats.

Morphological and Molecular Identification The cap of T. equestre is broadly convex with an inrolled margin in young specimens or nearly flat in older. The color of fruiting bodies is bright yellow to yellow‐green when immature, often with a brownish umbo. With aging, the color changes to olive‐green with a brown or brown‐red shade. The main pigment compound found in T. equestre is flavomannin‐6,6‐dimethylether (Steglich, Topfer, Reininger, Gluchoff, & Arpin, 1972). Caps of young specimens are sticky, and usually dry when matured. Cap diameter varies from 3 to 15 cm. Gills are emarginated, rather broad, medium spaced, pale chrome to pale yellow, with entire edges. Spores are white, elliptical, 5 to 8.5 × 3 to 6 μm. The stipe is usually yellow to yellow‐green 3 to 10 cm long, with an even diameter. Flesh is white to very pale yellow near the cap surface; not changing on exposure. The main volatile compound in T. equestre is 1‐octen‐3‐ol of woody aroma and methyl‐2‐phenyl with champignon mycelium odor (Woźniak, Sobkowska, & Kwiatkowska, 1983). Its fruiting season begins in late summer and autumn and lasts to the beginning of winter. T. equestre, T. flavovirens, and T. auratum had originally been considered as three separate species belonging to the Tricholoma genus, although all three share very similar morphological features and are very difficult to distinguish using macro‐ and microscopy methods. In Europe, two additional varieties of T. equestre have also been recognized by some mycologists: T. equestre var. populinum (Christensen & Noordeloos), associated with a deciduous habitat represented by Populus sp. and/or Betula sp. trees, and T. equestre var. pallidifolia characterized by pale to white gills, also sometimes identified as a representative of T. joachimii (Bon & Riva). Recent molecular analyses (based on the internal transcript spacer (ITS)1/5.8S/ITS2 region of the nuclear ribosomal unit and the 5′ part of the mitochondrial cox1 gene) support the complex phylogeny of T. equestre, and provide further evidence that T. equestre, T. flavovirens and T. auratum from various geographical regions (Europe, North America, and Asia) are all representatives of a single species and should formally be considered as T. equestre (Deng & Yao, 2005a; Horton, 2002; Kalamees, 2001; Moukha et al., 2013). It is also likely that a number of varieties and subspecies may occur in various geographical locations. Phylogenetic analyses of ITS patterns indicate that T. equestre represents a species complex which still remains poorly resolved (Heilmann‐Clausen, Christensen, Frøslev, & Kjøller, 2017). Moreover, the conducted investigations also support the view that what was identified previously as T. equestre var. pallidifolia (or T. joachimii) is a representative of other species not belonging to the T. equestre species complex while T. equestre var. populinum (along with some specimens from France identified earlier as T. equestre) belongs to the T. frondosae clade (Moukha et al., 2013). In summary, the findings strongly suggest the need for molecular analyses in correct T. equestre identification. Since such tools are now widely accessible, a genetic‐based taxonomic characterization of the investigated mushroom should be presented in any study involving T. equestre, including ecological, nutritional, biomedical and toxicological studies (including reports of poisoning, if mushroom material is available). A preferred approach is to perform PCR amplification of the rDNA ITS1/5.8/ITS2 region using primer pair ITS4/ITS5 (5′‐ GCATATCAATAAGCGGAGGA‐3′/5′‐ GGAAGTAAAAGTCGTAACAAGG‐3′) (White, Bruns, Lee, & Taylor, 1990) and amplification of the 5′ region of the mitochondrial cox1 gene using primer pair CoxU1/CoxR (5′‐TCTACTAATGCTAAAGATATTGG‐3′/5′‐ CACCGGCTAATACAGGTAA‐3′) (Damon et al., 2010).

Similar Species T. equestre (Christensen & Heilman‐Clausen, 2013 2010 Tricholoma genus that are characterized by yellow or green‐yellow caps and stipes (Figure (i) Tricholoma frondosae Kalames & Shchukin. Widespread in Europe (common from southern Sweden and Finland, Estonia to Northern Poland). Cap (diameter 5 to 11 cm), at first broadly conical, bell‐shaped to convex, when mature flattened with low, broad umbo. It is covered by distinct, appressed, concentrically arranged scales, denser in the central part. The color of the cap is very diverse: pale yellow, greenish yellow, mustard yellow, while scales are usually yellowish brown or olivaceous. Gills are emarginate, medium spaced to crowded, in different tints of yellow. Stipe is cylindrical, often bulbous at base, pale yellow to sulfur yellow. Length of the stipe in mature specimens from 5 to 13 cm. Spores are white and elliptical but smaller than those of T. equestre ‐ 4,5‐6,5 × 3,8‐4,5 μm. Flesh is white to slightly yellow with a mild farinaceous smell. Unlike T. equestre , T. frondosae occurs in nutrient and humus rich calcareous soils. It is associated with aspen ( Populus tremula ) and to a lesser extent with Picea and Abies . Fruiting from late July to October.

Kalames & Shchukin. Widespread in Europe (common from southern Sweden and Finland, Estonia to Northern Poland). Cap (diameter 5 to 11 cm), at first broadly conical, bell‐shaped to convex, when mature flattened with low, broad umbo. It is covered by distinct, appressed, concentrically arranged scales, denser in the central part. The color of the cap is very diverse: pale yellow, greenish yellow, mustard yellow, while scales are usually yellowish brown or olivaceous. Gills are emarginate, medium spaced to crowded, in different tints of yellow. Stipe is cylindrical, often bulbous at base, pale yellow to sulfur yellow. Length of the stipe in mature specimens from 5 to 13 cm. Spores are white and elliptical but smaller than those of ‐ 4,5‐6,5 × 3,8‐4,5 μm. Flesh is white to slightly yellow with a mild farinaceous smell. Unlike , occurs in nutrient and humus rich calcareous soils. It is associated with aspen ( ) and to a lesser extent with and . Fruiting from late July to October. (ii) Tricholoma sulphureum (Bull.: Fr.) P. Kumm. Widespread in Europe (except northern Scandinavia and Russia) and North America, also found in Asia (Deng & Yao, 2005b T. equestre , its cap is slightly smaller (diameter up to 9 cm) when young conical, bell‐shaped to convex, later low convex to flat, sometimes with a low umbo. The color of the cap is sulfur yellow, greenish yellow, lemon yellow, cinnamon to orange brown. Gills are adnate to deeply emarginate, thick, medium spaced. Gill color is similar to the cap, often more saturated. Stipe is cylindrical 3 to 11 cm. Stipe and flesh are yellow in color. Spores are white ellipsoidal, smooth, with a pronounced attachment peg. Spore size is 9 to 12 × 5 to 6.5 μm. T. sulphureum produces a characteristic unpleasant odor caused by the chemical skatole, described as a coal gas. This feature is often used to distinguish it from other Tricholoma species characterized by yellow caps. It is ectomycorrhizal mainly with deciduous broadleaf trees: oaks and beech but can also be found occasionally in coniferous habitats. Fruiting occurs from late summer to December.

(Bull.: Fr.) P. Kumm. Widespread in Europe (except northern Scandinavia and Russia) and North America, also found in Asia (Deng & Yao, , its cap is slightly smaller (diameter up to 9 cm) when young conical, bell‐shaped to convex, later low convex to flat, sometimes with a low umbo. The color of the cap is sulfur yellow, greenish yellow, lemon yellow, cinnamon to orange brown. Gills are adnate to deeply emarginate, thick, medium spaced. Gill color is similar to the cap, often more saturated. Stipe is cylindrical 3 to 11 cm. Stipe and flesh are yellow in color. Spores are white ellipsoidal, smooth, with a pronounced attachment peg. Spore size is 9 to 12 × 5 to 6.5 μm. produces a characteristic unpleasant odor caused by the chemical skatole, described as a coal gas. This feature is often used to distinguish it from other species characterized by yellow caps. It is ectomycorrhizal mainly with deciduous broadleaf trees: oaks and beech but can also be found occasionally in coniferous habitats. Fruiting occurs from late summer to December. (iii) Tricholoma sejunctum (Sowerby) Quél. It is abundant in North America and Europe (except far north) but can also be found in Japan, Korea and Costa Rica. Cap diameter is similar to that of T. sulphureum (5 to 9 cm). The cap at first is bell‐shaped to convex, when mature rather flat with a large broad umbo. Its cap color is yellowish to yellowish olive; with dark, radiating fibers. Gills are emarginate, broad, medium spaced and conversely to T. equestre , they are white, sometimes with a slight yellow tint. Stipe morphology is similar to that of T. equestre although they appear much more whitish, sometimes with a yellow tint or olivaceous flushes at the base. Flesh is white (only on top of the cap with a yellow tint) and does not change on exposure. Spores are smooth and elliptical (5 to 8.5 × 3 to 6 μm), white in color. T. sejunctum is ectomycorrhizal both with deciduous (hornbeams, oaks, beeches) and coniferous (pines and spruces) trees. Reported from August to November.

(Sowerby) Quél. It is abundant in North America and Europe (except far north) but can also be found in Japan, Korea and Costa Rica. Cap diameter is similar to that of (5 to 9 cm). The cap at first is bell‐shaped to convex, when mature rather flat with a large broad umbo. Its cap color is yellowish to yellowish olive; with dark, radiating fibers. Gills are emarginate, broad, medium spaced and conversely to , they are white, sometimes with a slight yellow tint. Stipe morphology is similar to that of although they appear much more whitish, sometimes with a yellow tint or olivaceous flushes at the base. Flesh is white (only on top of the cap with a yellow tint) and does not change on exposure. Spores are smooth and elliptical (5 to 8.5 × 3 to 6 μm), white in color. is ectomycorrhizal both with deciduous (hornbeams, oaks, beeches) and coniferous (pines and spruces) trees. Reported from August to November. (iv) Tricholoma joachimii Bon & A.Riva. It is a rare European species with a scattered distribution. More commonly found in southern parts of Europe. The cap diameter of mature specimen varies from of 5 to 12 cm and is convex to flattened, sometimes with a broad umbo. Its color is honey‐brown, to brownish‐olive; sometimes with a yellow tint, usually palest in the marginal zone. Brown scales occur in the central part of the cap. Gills are emarginate, medium broad and medium spaced, whitish to pale cream. Stipe is usually 4 to 8 cm long, cylindrical or sub‐bulbous. The upper part is whitish while the lower is often with brown or yellow discoloration. Flesh is white and farinaceous in smell. Spores are smooth and elliptical (6 to 8 × 5 to 6 μm) white. T. joachimii is a mycorrhizal fungus associated with Pinus spp. The distribution of this fungi seems to be restricted to semi‐open or xerothermic forests on sandy, mineral rich and calcareous soils. Fruiting from September to November.

Bon & A.Riva. It is a rare European species with a scattered distribution. More commonly found in southern parts of Europe. The cap diameter of mature specimen varies from of 5 to 12 cm and is convex to flattened, sometimes with a broad umbo. Its color is honey‐brown, to brownish‐olive; sometimes with a yellow tint, usually palest in the marginal zone. Brown scales occur in the central part of the cap. Gills are emarginate, medium broad and medium spaced, whitish to pale cream. Stipe is usually 4 to 8 cm long, cylindrical or sub‐bulbous. The upper part is whitish while the lower is often with brown or yellow discoloration. Flesh is white and farinaceous in smell. Spores are smooth and elliptical (6 to 8 × 5 to 6 μm) white. is a mycorrhizal fungus associated with spp. The distribution of this fungi seems to be restricted to semi‐open or xerothermic forests on sandy, mineral rich and calcareous soils. Fruiting from September to November. (v) Tricholoma aestuans (Fr.) Gillet. Found in North America and Europe (common only in boreal, mountainous habitats). Cap diameter of mature specimens varies from 2 to 7 cm, hemispheric to convex at first with margin inrolled, expanding to broadly convex or plane with a conical or obtuse umbo. Cap of young specimens is pale chrome to honey later olivaceous to yellow brown with brownish umbo. Gills almost free, in color pale yellow to yellow similar to T. equestre . Stipe 5 to 14 cm long, up to 2 cm thick equal or base clavate, sulfur‐yellow to lemon‐chrome. Spores 5 to 7 × 4.5 to 5.5 μm white, smooth and elliptic. Flesh of T. aestuans is white or pale yellow. T. aestuans is ectomycorrhizal mainly with coniferous trees ( Pinus and Picea ) and occurs mostly on nutrient poor sandy soils. The feature that easily distinguishes T. aestuans from T. equestre is its bitter flavor.

(Fr.) Gillet. Found in North America and Europe (common only in boreal, mountainous habitats). Cap diameter of mature specimens varies from 2 to 7 cm, hemispheric to convex at first with margin inrolled, expanding to broadly convex or plane with a conical or obtuse umbo. Cap of young specimens is pale chrome to honey later olivaceous to yellow brown with brownish umbo. Gills almost free, in color pale yellow to yellow similar to . Stipe 5 to 14 cm long, up to 2 cm thick equal or base clavate, sulfur‐yellow to lemon‐chrome. Spores 5 to 7 × 4.5 to 5.5 μm white, smooth and elliptic. Flesh of is white or pale yellow. is ectomycorrhizal mainly with coniferous trees ( and ) and occurs mostly on nutrient poor sandy soils. The feature that easily distinguishes from is its bitter flavor. (vi) Tricholoma arvense Bon. Widespread in Northern Europe (Fennoscandia, Denmark and Northern Russia) also found in Northern America. Mature cap diameter varies from 5 to 16 cm. When young the cap shape is broadly conical or convex, when mature convex to flattened with a large umbo with an undulating marginal zone, sometimes the central part of the cap is scaly. Young caps are yellow‐green to olivaceous‐green later honey, orange‐yellow to yellow‐brown. Gills are adnate to emarginate, medium spaced and unlike T. equestre white or with a gray tint. The stipe of mature specimens can vary from 4 to 12 cm, cylindrical, fibrillose, white in the upper part and pale yellow or brown in the basal. Spores 4 to 6 × 3.3 to 5 μm white, smooth and elliptic. Flesh of T. arvense is usually white or pale gray, after cutting at the basal end changing color into reddish. Smell is farinaceous, however taste is unpleasant—rancid. Typical for nutrient poor sandy soils, ectomycorrhizal with coniferous trees (in northern range mainly with Pinus sylvestris in Central Europe also with Abies. Fruiting from August to October. There are a number of mushroom species that share their distribution and some morphological features with(Christensen & Heilman‐Clausen,; Kibby,). The most likely reason for erroneous identification is associated with other species belonging thegenus that are characterized by yellow or green‐yellow caps and stipes (Figure 2 ). These species include: Figure 2 Open in figure viewer PowerPoint Morphology of fruiting bodies of (a) Tricholoma equestre, (b) Tricholoma joachimii, (c) Tricholoma sulphureum, (d) Tricholoma sejunctum, (e) Tricholoma frondosae, and (f) Tricholoma arvense. Species identified on basis of morphological features by a professional mycologist (Pictures by Ryszard Rutkowski). Due to its distinctively different morphological features, there is a lower chance of mistaking T. equestre for other gilled mushroom species with green or yellow green caps and/or stipe such as Rusulla aurea Pers., R. clavoflava Grove, young specimens of Amanita phalloides (Vaill. ex Fr.) Link and many others.

Nutritional Value Being valued for its taste, T. equestre has a long tradition of collection from the wild as food. Usually both caps and stipes, which can be dried, frozen or freshly prepared, are consumed in different forms: fried, boiled, soured or pickled. As noted by some traditional mushroom cookery books, T. equestre can be used to prepare a soup without prior blanching (Maćkiewicz & Falandysz, 2012). Carbohydrates, amounting to 35 to 60 g/100 g dry weight (dw), represent the most abundant macronutrients of T. equestre. It contains detectable levels of glucose (0.9 g/100 g dw) and has a relatively high content of polyol mannitol (8 g/100 g dw). Similarly to other mushrooms, T. equestre is also a relatively rich source of proteins (14 to 18 g/100 g dw) with albumins being the prevalent fraction (Florczak, Karmańska, & Wędzisz, 2004; Jedidi, Ayoub, Philippe, & Bouzouita, 2017). As reported by Ribeiro et al. (2008), the most abundant free indispensable amino acids are alanine (687 mg/100 g dw), lysine (252 mg/100 g dw), and leucine (102 mg/100 g dw). Like many other mushroom species, T. equestre has a low content of lipids, within a range of 2 to 7 g/100 g dw (Florczak et al., 2004; Jedidi et al., 2017). Compared to Boletus edulis Bull., it contains a slightly higher content of saturated fats (with palmitic, stearic and pentadecanoic acids being the major fraction), a 20‐fold higher content of monounsaturated fatty acids (particularly oleic acid) and a 26‐fold higher content of polyunsaturated fatty acids (particularly linoleic, arachidonic and γ‐linoleic acids) (Ribeiro, Pinhoa, Andradea, Baptistab, & Valentao, 2009). The estimated energy content is 1522 kJ/100 g dw (Jedidi et al., 2017). The content of vitamin B1 (thiamine) and B2 (riboflavin) falls in the range of 0.40 to 0.85 mg/100 g dw and 0.50 to 0.85 mg/100 g dw, respectively (Karosene & Vilimaite, 1971). The content of ergosterol (2.2 mg/100 g dw), a vitamin D2 precursor, is rather low in T. equestre when compared to other edible mushrooms (Carvalho et al., 2014). As found, it is usually rich in Na (Table 1). Noticeably, the mean Na content in T. equestre (2900 mg/kg dw) largely exceeds the range of 100 to 400 mg/kg dw, usually observed for wild mushroom species (Kalač, 2009). One should note that this species can grow on soils with high salinity, as noted for specimens collected from the Hel Peninsula in Poland that revealed a mean Na content in stipes reaching 11000 mg/kg dw (Maćkiewicz, Dryżałowska, Mielewska, & Falandysz, 2006). The mean content of Ca, Mg, Cu, and Mn in T. equestre falls within the usual ranges of minerals in wild mushroom species as reported by Kalač et al. (2009). The content of K, Fe, and Zn is higher than generally observed while the content of P and Se is lower (Table 1). A study using an artificial gastric fluid system has demonstrated a high bioavailability of Ca, Cu, and Mg from T. equestre fruiting bodies (Kała et al., 2017). Table 1. Element content (mg kg−1 dry weight) observed in fruiting bodies of Tricholoma equestre Element Mean Min Max SD References Macroelements Ca 269.4 31 780 268.4 b, e, f, g, p, x K 43801 14000 83000 21623.6 b, c, e, f, g, p, x Mg 1144.2 600 2414 602.7 b, e, f, g, p Na 2900 60 11000 3229.2 b, c, e, f, g, p P 4685 2500 8300 1984.4 b, e, f, g Trace elements Ag 1.88 0.48 3.5 1.2 a, e, f, g Al 345.7 70 943 326.9 a, b, e, g, o As 0.33 0.08 a Au 0.02 0.01 a B 0.3 0.1 a Ba 4.1 0.35 19.1 6.5 a, b, e, g, o Bi 0.02 0.01 a Cd 1.33 0.26 2.1 0.55 a, b, d, e, f, g, i, m, t, w Co 0.92 0.07 4.1 1.38 a,,b, e, g, j, k Cr 2.7 0.08 13.7 4.77 a, b, d, e, g, o Cs 22.97 9.5 39.1 14.98 b, c, o Cu 36.2 15.9 55 11.27 a, b, d, e, f, g, n, p, u, x Fe 247.2 4.9 632 226.73 a, b, d, e, f, g, h, p, x Ga 0.81 0.02 1.6 1.12 a, n Ge 0.01 0.01 a Hg 0.95 0.12 3.3 0.84 k, l, r In 0.06 0.02 a Li 0.09 0.02 a Mn 34.11 8.4 100.4 27.65 a, b, d, e, f, g, h, x Nb 13.2 o Ni 2.43 0.12 11.8 4.23 a, b, d, e, f, g Pb 1.64 0.44 4 1.16 a, b, d, e, f, m, o, s, w, x Rb 696.1 88.2 2000 576.2 b, c, e, f, g, n, x Re 0.21 0.05 a Sb 0.09 0.02 a Se 0.45 0.11 a Sr 3 0.09 17.7 5.77 a, b, e, f, g, n Te 0.27 0.07 a Th 1.17 0.042 2.3 1.59 b, o Tl 0.21 0.05 a U 2.6 0.01 5.2 3.67 b, o V 10.7 o Zn 182.1 16.2 460 104.8 a, b, d, e,f, g, p, u, x Zr 66.3 o Rare earth elements Ce 5.9 0.24 17.3 9.8 a, n, o Er 1.5 n Gd 0.03 0.01 b, n La 0.14 0.12 0.16 0.028 b, n Lu 0.01 n Nd 5.26 0.12 10.4 7.12 n. o Pr 0.02 n Sm 0.025 0.02 0.03 0.007 b. n Tb 0.03 n Tm 0.02 n Y 0.03 n Yb 0.01 n Platinum group elements Ir 0.02 n Os 0.02 n Pd 0.02 n Rh 0.02 n Ru 0.02 n Pt 0.02 n Qualitatively, the profile of organic acids in T. equestre is similar to that of B. edulis but their total content is higher (94.0 to 99.3 g/kg dw). The determined acids include oxalic (2.1 to 2.6 g/kg dw), aconitic (4.6 to 5.2 g/kg dw), citric (22.0 to 23.7 g/kg dw), 57.4 to 61.1 g/kg dw), and fumaric (6.7 to 7.9 g/kg dw). A phenolic compound p‐hydroxybenzoic acid (35.5 mg/kg dw) has also been determined in T. equestre (Ribeiro et al., 2006). Moreover, compared to other mushrooms, T. equestre can be a rich source of β‐carotene, particularly in its caps. Lycopene, a precursor of its biosynthesis, can also be detected, at higher levels in stipes (Robaszkiewicz, Bartosz, Ławrynowicz, & Soszyński, 2010). In spite of this, T. equestre displayed rather low antioxidant capacity as found using 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH) free radical and 2,2′‐azino‐bis‐3‐ethylbenzthiazoline‐6‐sulfonic acid (ABTS) assays (Ribeiro et al., 2006; Robaszkiewicz et al., 2010). The flavomannin‐6,6‐dimethylether, a polyphenol with a dimeric pre‐anthraquinone structure that is thought to be a mushroom yellow pigment, has been isolated and purified from cooked fruiting bodies, and further demonstrated to exhibit in vitro cytostatic effects in human adenocarcinoma colorectal Caco‐2 cells by inducing cell‐cycle arrest without genotoxic activity (Pachón‐Peña et al., 2009; Steglich et al., 1972). A study by Muszyńska, Sułkowska‐Ziaja, and Ekiert (2009) reported that T. equestre fruiting bodies contain indoles such as tryptamine (2.0 mg/100 g dw) and serotonin (0.18 mg/100 g dw). Unsurprisingly, the work also demonstrated the presence of serotonin‐precursor, tryptophan, at 2 mg/100 g dw, which is consistent with observations made by Ribeiro et al. (2008). However, Muszyńska et al. (2009) concluded that due to the toxicity of this amino acid and the neuro‐transmitting activity of serotonin a T. equestre cannot be considered as safe for human consumption. This conclusion should be disregarded as tryptophan is one of the indispensable amino acids, commonly found in foodstuffs and its recommended daily intake has been set at 4 mg/kg body weight (National Academy of Sciences, 2005). In other words, daily consumption of as much as 1 kg of fresh mushrooms by a 60‐kg adult would constitute just 1% of this guideline level. Serotonin, in turn, is commonly found in various foodstuffs, also at levels higher than those of T. equestre (for example, in bananas) although its dietary intake has no physiological effect as it cannot cross a brain‐border (Feldman, Lee, & Castleberry, 1987; Young et al., 2007).

Contamination Level The ability of mushrooms to uptake and accumulate a number of environmental contaminants is well established, as extensively shown by numerous field studies and experimental data (Kalač & Svoboda, 2000; Rzymski, Mleczek, Siwulski, Gąsecka, & Niedzielski, 2016). Nevertheless, this is highly dependent on environmental quality and can vary between species (Kalač, 2013; Mleczek et al., 2016a). The majority of studies assessing contamination of wild‐mushrooms, including T. equestre, have employed various spectroanalytical methods (for example, optical emission spectrometry, mass spectrometry, X‐ray fluorescence) to determine the content of (potentially) toxic metals and metalloids. The summary of available data on elemental content observed in T. equestre fruiting bodies is summarized in Table 1. Information on the presence of organic pollutants (for example, polychlorinated biphenyls, dioxins) is missing. No study on T. equestre contamination has so far acquired the molecular tools to identify the phylogenetic position of the investigated mushroom specimens. As indicated by values of the bioconcentrated factor (BCF) calculated on basis of element content in soil, fruiting bodies of T. equestre significantly accumulate (BCF > 1) Cu, Se, and Zn (which is a common observation in aboveground mushroom species) and Ag, Cd, Re, and Tl (Alonso et al., 2003; Mleczek et al., 2016a). The juxtaposition of expected mean content in a standard serving of selected metals in T. equestre which are known to exert toxic effects at certain exposure levels (calculated on values given in Table 1) with established values of the Provisional Tolerable Weekly Intake (PTWI), Provisional Maximum Tolerable Daily Intake (PMTDI), Tolerable Daily Intake (TDI) or oral Permitted Daily Exposure (PDE) is presented in Table 2. The consumption of a single meal of 30 g dw fruiting bodies (equivalent of 300 g fresh biomass) would not contribute significantly to any of these guideline levels. This threshold would also not be exceeded even in the very unlikely scenario of repeated daily consumption of 30 g dw of T. equestre for 7 consecutive days. Table 2. Expected intakes of various elements considering the consumption of single serving of 30 g of dried Tricholoma equestre mushrooms by a 60‐kg adult calculated on means presented in Table , and the comparison to existing recommendations: Provisional Tolerable Weekly Intake (PTWI), Provisional Maximum Tolerable Daily Intake (PTMDI), Tolerable Daily Intake (TDI) or oral Permitted Daily Exposure (PDE) Element Expected mean content in standard serving [mg] PTWI [mg kg−1 bodyweight] PTWI for 60‐kg adult [mg] % PTWI for single consumption As 0.01 0.015 0.9 1.1 Al 10.4 2.0 120 8.6 Cd 0.04 0.0058 0.348 11.5 Hg 0.03 0.004 0.24 12.5 Pb 0.05 0.025 1.5 3.3 Element Expected mean content in standard serving [mg] PMTDI/TDI [mg kg−1 bodyweight] PMTDI/TDI for 60‐kg adult [mg] % PMTDI/TDI for single consumption Cra 0.08 0.3 18 0.45 Cu 1.1 0.5 30 4.0 Fe 7.6 0.8 48 15.8 Ni 0.07 0.0028 0.168 43.4 Zn 5.5 0.3‐1.0 18‐60 9‐30 Element Expected mean content in standard serving [mg] PDE [mg kg−1 bodyweight] PDE for 60‐kg adult [mg] % PDE for single consumption Pt 0.006 0.0026 0.156 3.8 An emerging group of pollutants is represented by rare earth elements (REEs). It consists of nine elements (La, Ce, Eu, Gd, Nd, Pr, Pm, Sm, and Sc) categorized as light REEs, and 8 elements (Dy, Er, Ho, Lu, Tb, Tm, Y, and Yb) representing heavy REEs. Numerous applications of REEs in the medical, industrial, and agricultural sectors have been developed over recent decades resulting in their increasing environmental levels (Pagano et al., 2015; Poniedziałek et al., 2017). The data concerning their toxicity is scarce and mostly limited to Ce, La, Nd, and Gd. Generally, the content of REEs in mushrooms is still little known (Falandysz, Sapkota, Mędyk, & Feng, 2017), and to date only 2 studies have considered T. equestre (Mleczek et al., 2016a, b). The total mean content of REEs observed in T. equestre amounts to 13.0 mg/kg dw and exceeds the maximum threshold (0.7 mg kg−1 fresh weight, equivalent to 7.0 mg/kg dw, assuming 90% moisture) set in China, so far the only country to regulate REEs in foodstuffs (SAC 2012). However, one should note that this result is influenced by the high content of Ce and Nd (17.3 and 10.4 mg/kg dw) found by Campos and Tejera (2011) in specimens collected in Spain from soils characterized by a relatively high content of these elements when compared to their average content observed for this country (Ramos et al., 2016). Moreover, the mushroom contents of Ce and Nd observed by Campos and Tejera (2011) were higher by an order and 2 orders of magnitude, respectively, than those reported for T. equestre collected in Poland by Mleczek et al. (2016a, b), and later also observed at a similar range in Macrolepiota procera (Scop.) Singer (Falandysz et al., 2017). Total levels of REEs observed by Mleczek et al. (2016a, b) were also lower than those reported recently for commercially cultivated mushrooms (for example, Pleurotus ostreatus (Jacq. ex Fr.) P.Kumm., Agaricus bisporus (J.E.Lange) Imbach) (Mleczek et al., 2018; Rzymski et al., 2017; Siwulski et al., 2017), and remained far below the guideline level implemented in China. Apart from the potentially different abiotic levels of REEs, the conflicting observations made by Campos and Tejera (2011) and Mleczek et al. (2016b) may also arise from the use of different analytical methods: X‐ray fluorescence spectrometry not validated on certified material (Campos & Tejera, 2011) and inductively coupled plasma optical emission spectrometry validated over five different certified materials (Mleczek et al., 2016b). Therefore, the potential interference of matrix cannot be excluded in the case of the Spanish mushrooms. Considering that T. equestre is collected from locations with no increased ambient contents of REEs, a significant contribution of this mushroom in their dietary intake is rather unlikely.

In Vivo Toxicity In vivo toxicology, in which whole foods or their ingredients are administrated to animals for evaluation of acute, subacute, or chronic effects, has been a gold standard in toxicity assessment. The majority of animals employed for such purpose are rodents, mostly mice, rats or rabbits. In the case of mushrooms, an in vivo toxicological assessment has almost always been employed after documented poisonings in humans. Prior to a report by Bedry et al. (2001) on an outbreak of cases of rhabdomyolysis following T. equestre consumption, not a single study had evaluated the toxicity of this species, considered traditionally as edible, in any experimental model. A first in vivo study was actually performed by Bedry et al. (2001) in order to evaluate whether T. equestre could potentially be a causative agent of observed human poisonings. Adult male Swiss mice (each group n = 3) were given a powder of freeze‐dried fresh T. equestre for 3 days by gastric intubation in three doses: 2, 4, and 6 g/kg body weight (bw). Treatment with the latter 2 doses led to a significant increase in serum creatine kinase 48 hr from the last exposure, from 145 ± 40 U/L (control group) to 345 ± 120 and 380 ± 25 U/L, respectively. In the second trial, mice (n = 5) were given a total dose of 6 g/kg bw in the form of aqueous (cold and boiled), chloroform‐methanol and lipid‐free chloroform‐methanol extracts once a day by gavage for 3 days. The results were compared to extracts obtained in the same manner from P. ostreatus and 70 mg/kg bw/day p‐phenylenediamine, serving as a positive control. A significant increase in serum creatine kinase concentration was noted after 96 hr from last dosage in groups treated with boiled and chloroform‐methanol lipid free extracts of T. equestre—it amounted to 912 ± 425 and 883 ± 500 U/L, respectively, but was at least two‐fold lower than levels observed for the group treated with p‐phenylenediamine (1828±450 U/L) (Bedry et al., 2001). Another contribution to an in vivo assessment of T. equestre toxicity was made by 2 studies of Nieminen et al. (2005, 2008). In both studies, mushroom specimens were collected from mixed Picea and deciduous forest from locations in which no significant contamination could be expected. The first study evaluated the effects in an unspecified laboratory strain of female Mus musculus L. mice from the breeding colony of the Univ. of Joensuu, Finland. The animals (each group n = 6) were given dried powder of T. equestre at 3, 6, and 9 g/kg bw/day or freshly frozen mushrooms at 9 g/kg bw/day for 5 consecutive days. Contrary to Bedry et al. (2001) who performed gavage administration, this study used mushrooms mixed into the feed of the animals. After the treatment period, an increase in creatine kinase concentration was noted only in the group receiving 9 g/kg bw/day of dried T. equestre (1171±313 U/L compared to 777 ± 157 U/L observed in the group treated with 70 mg/kg bw/day of p‐phenylenediamine). The same dose of freshly frozen mushrooms did not affect this parameter significantly. No changes in aspartate and alanine aminotransferase were noted for any treatment group. All animals remained in good health. The interpretation of these results was complicated by the simultaneous observation that dried Boletus edulis (used as a reference mushroom of well‐established edibility, similarly to P. ostreatus in Bedry et al. (2001) also caused a significant increase in creatine kinase concentration at 9 g/kg bw/day. In the second study, Nieminen et al. (2008) evaluated the subchronic hepato‐, cardio‐ and myotoxicity of freshly frozen T. equestre mixed with normal animal feed and given to male NIH/S mice (each group n = 6) at 12 g/kg bw/day for 4 weeks. Compared to an unexposed control group, a statistically significant increase in concentrations of total creatine kinase (446 ± 21 U/L compared with 307±75 U/L) and in its MB fraction (427±39 U/L compared with 292 ± 62 U/L) was observed. This was the only in vivo study to additionally assess the MB fraction of creatine kinase. No changes in aminotransferase activity were observed. The histological evaluation of dissected tissue samples revealed a higher frequency of inflammatory state in the pericardial fat for the group to which T. equestre was administrated. No histological alterations in muscle and kidney were observed while changes in the liver occurred at the same frequency between the treated and control group. All animals remained in good health. Contrary to the findings of Bedry et al. (2001) and Nieminen et al. (2005, 2008), a study conducted by Chodorowski et al. (2004) on male BALB/c mice (n = 5 in each group) did not find any significant effect of freeze‐dried powder of T. equestre nor its boiled aqueous and chloroform‐methanol extracts (all given by gavage at dose of 12g/kg bw/day for 3 days) on serum creatine kinase concentration measured 72 hr after a final dose (157 ± 93, 129 ± 30, and 96 ± 38 U/L, respectively, compared to 107 ± 38 U/L in control). However, this study used mushrooms that had been frozen at –20 °C for 1 year before being given to the tested animals. In turn, a p‐phenylenediamine (70 mg/kg bw/day), used as a positive control, caused a significant increase in enzymatic activity (265 ± 63 U/L). All animals survived the experiment. In all in vivo toxicological studies on T. equestre to date, mushroom specimens were recognized on the basis of their morphological features not molecular analyses. With no genetic phylogeny available it is not known whether these studies employed comparable T. equestre strains, and the use of representatives of other clades than the T. equestre complex cannot fully be excluded. According to Bedry et al. (2001), the mushrooms were collected in southwestern France – one should note that recent molecular investigations have revealed that some French specimens, previously considered as T. equestre, in fact belong to the distinct T. frondosae clade (for details see the “Morphological and molecular identification” section). These mushrooms are extremely difficult to distinguish by morphology, even for a qualified mycologist. One should also note a number of limitations associated with the in vivo model used to test the myotoxicity of T. equestre extracts. Firstly, the doses at which significant effects were detectable (mostly by increased creatine kinase concentration) were extremely high. In the case of the study of Bedry et al. (2001), the most significant changes were elicited at doses corresponding to 3 kg of fresh mushrooms consumed daily for 3 consecutive days by a 60‐kg adult. In the investigations of Nieminen et al. (2005, 2008), the effects were noted at doses equivalent to 4 kg of fresh T. equestre eaten every day for 5 days by a 60‐kg individual. Such consumption by a human is virtually impossible. Moreover, at lower but still relatively high doses (up to nearly 3 kg eaten every day) no significant effects were recorded in mice (Nieminen et al., 2005, 2008). It is also known that pre‐analytical conditions such as handling of the animal during blood collection and site of sampling can significantly affect the determined creatine kinase concentrations (Matsuzawa & Ishikawa, 1993). Moreover, rodents should be accustomed to laboratory handling procedures (for example, venipuncture without blood withdrawal) prior to experiments during which samples are collected for determination of creatine kinase concentration, as otherwise repetitive blood sampling may cause a significant increase in its level independently to that resulting from muscle damage (Lefebvre, Jaeg, Rico, Toutain, & Braun, 1992). Unfortunately, none of the in vivo studies performed on T. equestre offer any reassuring information that factors artificially influencing creatine kinase activity had been ruled out. Instead, all studies present a rather high variation of obtained results in treated groups, as indicated by values of standard deviation. As noted by Nieminen, Mustonen, Kirsi, and Kärjä (2009a) in the brief summary on their in vivo studies, even in the group treated with the highest dose (12 g/kg bw/day for 28 days), some animals revealed unelevated creatine kinase concentration. Finally, it should be outlined that the effects reported for laboratory mice after ingestion of high doses of T. equestre may equally represent an unspecific response. A number of mushroom species classified as edible and traditionally consumed in various geographical regions have also been observed to adversely influence biochemical markers in rodents. Agaricus bisporus has been found to increase plasma bilirubin concentration while Lentinula edodes (Berk.) Pegler, Cantharellus cibarius Fr., Albatrellus ovinus (Schaeff.) Kotl. & Pouzar, Leccinum versipelle (Fr. & Hök) Snell and Imleria badia Fr. have all increased plasma creatine kinase activity in mice at 9 g/kg bw/day administered over 5 consecutive days to levels comparable to that observed in animals treated with similar doses of T. equestre (Nieminen et al., 2005, 2006; Nieminen, Kärjä, & Mustonen, 2009b). More recently, oral administration of 6 g/kg bw/day of Flammulina velutipes (Curtis) Singer has also been found to increase plasma concentration of total creatine kinase and its MB isoenzyme (Mustonen et al., 2018). Importantly, regardless of dosage and tested mushroom species only a modest increase in creatine kinase levels (up to several hundred U/L) was observed, particularly when compared to results observed in dystrophic mice or those treated with agents known to induce rhabdomyolsysis (up to few thousand U/L) (Osaki et al., 2015; van Putten et al., 2012). The findings of in vivo experiments could well support a hypothesis that laboratory mice may be sensitive to a mushroom‐based diet or that various edible mushrooms may cause adverse effects if consumed in great amounts. Surprisingly, the study of Chodorowski et al. (2004) did not find any effects of powder/extracts made from T. equestre in BALB/c mice. This may potentially be due to the conditions under which the mushrooms were stored prior to the experiments (–20 °C for 1 year) or by intraspecific differences between laboratory mice strains. A second hypothesis can be partially supported by the effects exerted by p‐phenylenediamine in BALB/c mice which, although significant, were several‐fold lower than those observed by Bedry et al. (2001) and Nieminen et al. (2005). In other words, BALB/c mice may be less responsive to myotoxic agents than other laboratory strains. Considering that the extensive consumption of any food has its own risks, we question whether the in vivo findings are meaningful enough to classify any of abovementioned mushrooms, including T. equestre, as inedible or even potentially toxic.

Human Subject Research Data from interventional studies involving human subjects consuming T. equestre is limited to only two studies. Nieminen et al., 2005 recruited four healthy volunteers who consumed a single portion of 150 mg of dried T. equestre per kg bw, an equivalent to approximately 100 g of fresh mushrooms consumed by a 70 kg adult. The specimens for investigation were collected in mixed Picea and deciduous forest in Finland, from a location far from heavy road traffic or industrial activities. The concentrations of plasma creatine kinase, creatine, aspartate, and alanine aminotransferase, glucose and lipid concentrations were monitored 3 and 7 days after consumption, and compared to a baseline level. No significant change in any parameter was observed (Nieminen et al., 2005). A larger study was conducted by Chodorowski et al. (2005) who monitored biochemical parameters in 56 subjects (30 females, 26 male) aged 18 and 76 years old voluntarily consuming T. equestre as a single meal of 70 and 150 g of fresh mushrooms (n = 43) or for 4 consecutive days at a total dose ranging from 300 and 1200 g. Over half (57.1%) of the investigated subjects suffered from type 2 diabetes, 48.2% took statins (simvastatin, lovastatin, fluvastatin, and atorvastatin) and 12.5% were using fibrates (enofibrate, ciprofibrate) to treat hyperlipidemia. There was no statistically significant increase in concentration of serum creatine kinase, aspartate and alanine aminotransferase in any studied individual 3 to 6 days after the last mushroom meal. These findings are meaningful considering that statins themselves can induce rhadbomyolysis (Mendes, Robles, & Mathur, 2014) while their effect may be potentiated by other compounds (for example, fibrates, macrolides), particularly those interacting with statin metabolism (Bellosta, Paoletti, & Corsini, 2004). It appears that T. equestre may not cause a similar effect, although considering the presence of individual differences in statin sensitivity among the human population, some caution is necessary when formulating such a conclusion. The studies of Nieminen et al. (2005) and Chodorowski et al. (2005) did not employ molecular tools for identification of collected specimens and assessment of their phylogenetic position. No survey on the frequency of adverse effects following consumption of T. equestre has so far been conducted on mushroom foragers from any location.

Clinical Cases of Poisoning A summary of human poisonings with T. equestre and observed symptoms is given in Table 3. All intoxications described in scientific literature are so far limited to regions of France, Poland, Germany and Lithuania. A total of 21 cases which involved rhabdomyolysis (without renal injury) have been documented. Mortality rate was 23.8% (5/21). Unfortunately, case descriptions rarely provide exact information on the applied treatment. Table 3. Cases of human poisonings linked with Tricholoma equestre consumption and manifested by rhabdomyolysis onset Region Sex Age Dose Time from last meal to symptoms onset [hr] Clinical symptoms CK max [U/L] AST max /ALT max [U/L] Recovery time [days] Reference France ♀ (7) ♂ (5) 22‐61 Not given 24‐72 Leg muscle weakness and myalgia, fatigue, facial erythema, profuse sweating. EMG: proximal thigh muscles without peripheral‐nerve involvement. Three cases were fatal (with myocardial lesion or renal injury) 226067 (♀) 34786 (♂) 1173/325 (♀) 8104/1392 (♂) Up to 15 days; death in 3 cases Bedry et al., 2001 Poland ♀ 48 100‐300g daily for 9 days 48 Leg muscle weakness and myalgia, profuse sweating without fever, nausea without vomiting 18150 802/446 21 Chodorowski et al., 2002 Poland ♂ 20 100‐300g daily for 9 days 48136 (CK‐MB: 888) 2002/454 Poland ♂ 5 300‐400 g daily for 4 days 4 Deep coma, cyanosis, convulsions. Respiratory failure. Positive Babiński sign. Muscle weakness (particularly the pelvic girdle and urinary bladder). Inability to walk 306 39/56 12 Chodorowski et al., 2003 Poland ♂ 72 300‐400g daily for 10 days 24 Leg muscle weakness and myalgia. Muscle weakness in area of chest, shoulders and abdomen. Respiratory failure. Fatal cardiac arrest 44767 1894/490 Death Anand et al., 2009 Germany ♂ 71 Unclear (a meal twice a day for 6 days) Unknown Muscle weakness and myalgia, profuse sweating, fatigue 4934 330/209 10 Horn et al., 2005 Lithuania ♂ 56 Unclear (1L of boiled mushrooms eaten 3 times a day for 5 days) 24 Leg muscle weakness, profuse sweating without fever, nausea, appetite loss. Erythorocyturia. ECG: repolarization disturbance, prolonged QT, left anterior fascicular block 30571 1190/457 19 Laubner & Mikulevičienė, 2016 Lithuania ♂ 73 Unclear 48 Leg muscle weakness and myalgia, profuse sweating without fever. Erythorocyturia. ECG: repolarization disturbance, prolonged QT 8011 408/154 14 Lithuania ♀ 55 Unclear 48 Fatigue, nausea, discomfort in the chest area. ECG: prolonged QT,: hemical changes in lateral, inferior, and interseptal myocardium wall 5363 454/244 12 Lithuania ♂ 44 Unclear (standard portion 3 time a day for 3 days) 24 Muscle weakness and myalgia, profuse sweating without fever, fatigue. Erythorocyturia, leukocytosis. ECG: prolonged QT, left anterior fascicular block, subpericardial injury. Heart attack. 34600 1524/579 Death Historically, the first documented poisoning cases to suspect T. equestre mushrooms as a causative factor were reported in 2001 (Bedry et al., 2001) and published as a brief report in the New England Journal of Medicine. Overall, it described 12 cases (7 women and 5 men aged 24 and 61) hospitalized between 1992 and 2000 with severe rhabdomyolysis approximately 1 week following at least three consecutive meals with T. equestre fruiting bodies collected in southwest France (Aquitaine region) (Bedry & Gromb, 2009). Three cases had a fatal outcome. As self‐reported by patients, an onset myalgia in the upper part of the legs occurred 25 to 72 hr after their last mushroom meal and worsened over the next 4 days causing stiffness. Moreover, facial erythema, nausea, profuse sweating and hyperpnea was noted for selected patients. Individuals with a fatal outcome also developed dyspnea, acute myocarditis (arrhythmia, cardiovascular collapse, and wide QRS complex) and hyperthermia. All 12 patients had elevated aspartate and alanine aminotransferases. Significantly increased serum creatine kinase and dark urine indicated rhabdomyolysis. In selected patients acute muscle injury was evidenced by electromyography and/or histological analyses of quadriceps femoris. Additionally, for three cases with a fatal outcome, myopathies were confirmed in psoas, arms, myocardium and diaphragm. No renal or hepatic failure was present. The clinical state of 9 patients was successfully normalized over the next 15 days, although muscular weakness was present for several weeks following hospitalization. Although the report of Bedry et al. (2001) ruled out the presence of any etiological factors of rhabdomyolysis in intoxicated patients (substance abuse, use of selected medications, occurrence of selected virus infections and parasites, and active systemic disorder), a number of unanswered questions remained. The dose of consumed T. equestre fruiting bodies was not estimated nor was the form of consumption established (fresh or dried; fried, boiled or as a soup). It is unknown if mushrooms were stored before consumption, and if so—under which conditions. This information is relevant if one considers that in all cases mushrooms were consumed as at least three consecutive meals (in other words, consumption over at least 3 days can be expected) while inappropriate storage (for example, prolonged room temperature, repeated freezing and thawing) may affect mushroom quality (Burton & Noble, 1993; Venturini, Reyes, Rivera, Oria, & Blanco, 2011). As shown for Tricholoma species, low activity of polyphenol oxidase prevents rapid browning of fruiting bodies when stored at 12 °C. although it does cause an increase in the activity of laccase that performs 1‐electron oxidations of polyphenols (Meihua & Yang, 2011), a process significantly contributing to the generation of reactive oxygen species and potentially further deterioration of chemical content (Wei et al., 2010). It should be noted that molds are frequently detected in mushrooms, including those of the Tricholoma genus, and some of those molds are known to be early decomposers of dead fruiting bodies (Brabcová, Nováková, Davidová, & Baldrian, 2016; Oh, Kim, Eimes, & Lim, 2018). It remains to be studied whether and how rapidly potentially toxinogenic mold species can colonize dead fruiting bodies of T. equestre. It is also unknown whether in cases of the poisonings reported by Bedry et al., 2001, mushrooms were eaten only by affected subjects or also by other individuals. The latter can be expected, and the fate of potential co‐consumers may be an informative clue when establishing to what extent individual susceptibility is involved in the development of clinical symptoms. Strikingly, the report by Bedry et al., 2001 provides no objective confirmation that T. equestre was actually consumed by the described subjects (for example, identification of Tricholoma spores in gastric content). The only evidence for concluding that T. equestre triggered rhabdomyolysis was based upon the in vivo experiments which were discussed in the previous section of this paper. To date no other cases of poisoning with T. equestre have been reported in France, although one should note that since 2004 it has been officially classified as toxic in this country (Bedry & Gromb, 2009). Another series of poisonings with T. equestre as a suspected cause were recently reported in Lithuania (Laubner & Mikulevičienė, 2016). They consist of four cases that occurred between 2004 and 2013, manifested by rhabdomyolysis with elevated creatine kinase concentration, accompanied by muscle pain, fatigue, nausea without vomiting and muscle pain, profuse sweating without fever, and respiratory insufficiency. Levels of aspartate and alanine aminotransferases were also significantly increased. Similarly to other poisoning cases associated with T. equestre and involving rhabdomyolysis, no renal insufficiency was noted. Electrocardiogram revealed myocardial repolarization disturbances. In one case, a patient died following a heart attack occurring 6 days after mushroom consumption (Laubner & Mikulevičienė, 2016). The description of poisoning cases has some notable shortcomings. Firstly, the exact dose of the ingested mushrooms remains unclear as it is reported as an undefined “standard meal” or as 0.5 to 1.0 L of boiled mushrooms consumed over a prolonged time (days, week, or month). It is also unknown whether the possibility of a mushroom being wrongly identified was ruled out. No data is given on mushroom storage conditions during the period they were consumed, relevant information particularly in the case of a patient who had consumed mushrooms once‐twice a day for 1 month. In two cases individual vulnerability can be suspected because co‐consuming people did not experience any symptoms following mushroom ingestion. In the other fatal case, a subject had a history of alcoholism, which itself can be a cause of rhabdomyolysis (Zimmerman & Shen, 2013). Information on previous experience (if any) in consuming T. equestre by the affected individuals is unfortunately lacking. One poisoning case involving T. equestre was also reported in Germany. A 71‐old‐year man was admitted to hospital after he consumed a mushroom meal twice a day for 6 consecutive days and observed muscle weakness and myalgia. As found, creatine kinase and aminotransferase concentrations were significantly increased, so were myoglobin levels. As self‐reported, the patient had often consumed large quantities of T. equestre in the past without any noticeable adverse effects. A poisoning indicating rhabdomyolysis occurred during a period in which the patient was using simvastatin. When hospitalized simvastatin treatment was discontinued and the patient received alkaline diuresis to prevent myoglobin precipitation in renal tubules (Horn, Prasa, Rothvinchow, & Hentschek, 2005). This is the only case report in which interactions between mushrooms and statins can be suspected, yet its exact mechanisms remain unknown. Statins themselves can cause rhabdomyolysis although it is more commonly reported when statins are used in conjunction with other drugs, which can potentiate an effect (Mendes et al., 2014; Thompson, Clarkson, & Karas, 2003). In this particular case of poisoning, it appears that T. equestre consumption could be a triggering factor (Horn et al., 2005). A series of poisonings with T. equestre, encompassing a total of 3 affected adult subjects and 1 child, were also recorded between 2001 and 2010 by two Polish medical units located in Gdańsk (Northern Poland) and Biała Podlaska (Eastern Poland). In all cases adverse effects had onset following consecutive ingestion of 100 to 400 g daily. The main clinical symptoms in adults included muscle weakness, nausea without vomiting, and significantly increased levels of creatine kinase, aspartate aminotransferase and alanine aminotransferase. One subject revealed an increased concentration of MB isoform of creatine kinase, and respiratory failure followed by cardiac arrest, eventually resulting in a fatal outcome. This case is the only one of all in documented T. equestre poisonings in which concentration of MB isoform of creatine kinase, a cardiac marker expressed mostly in the myocardium (Karras & Kane, 2001), was reported additionally to total creatine kinase level (Anand, Chwaluk, & Sut, 2009; Chodorowski, Waldman, & Sein Anand, 2002). A clinical course of poisoning in a 5‐year‐old child was distinctively different, with a rapid onset (4 hr after last mushroom meal) of deep coma, cyanosis and convulsions, although muscle weakness and increased creatine kinase was also observed (Anand et al., 2009; Chodorowski, Anand, & Grass, 2003). Similarly to the description of cases from France and Lithuania, some important information on the circumstances associated with T. equestre poisoning is missing. It is unknown whether the poisoned subjects had any previous exposure (and of what kind) to T. equestre. Information on sites of mushroom collection, conditions of storage during period of consumption and form in which they were prepared for consumption is also essentially lacking. The method used to confirm actual ingestion of T. equestre was not reported. It is likely that, similarly to the case reports, the source of confirmation was a self‐report of the poisoned patient, maybe also coupled with spore identification in gastric content. One should note, however, that distinction of Tricholoma species based on spore morphology, particularly isolated from such material, is very difficult if even possible at all (see ‘Similar species’ section for more details). An interesting statistic was provided by Gawlikowski, Romek, and Satora (2015) who summarized all mushroom poisoning cases recorded in 2002 to 2009 by a toxicological unit in Kraków, Poland. A total of 21 cases of poisoning with T. equestre were confirmed on the basis of spore identification in gastric fluids—the reported clinical symptoms included vomiting, abdominal pain and diarrhea. None of these cases were characterized by altered biochemical parameters (including creatine kinase activity) and no rhabdomyolysis was observed. Within the studied period, other mushroom species whose edibility is well established (for example, Armillaria mellea (Vahl) P. Kumm., Macrolepiota procera, Imleria badia or Suillus luteus (L.) Roussel), were a more common cause of such gastrointestinal events reported to the toxicological unit. The authors speculated that the most plausible cause of these effects was the inappropriate processing of mushrooms during transport and storage (Gawlikowski et al., 2015). In spite of the fact that the first cases of human poisoning with T. equestre were documented in scientific literature over 15 years ago, as yet no causative toxin has been identified and isolated. Instead, evidence for causing rhabdomyolysis has been characterized in other mushrooms such as Russula subnigricans Hongo (cycloprop‐2‐ene carboxylic acid) or Tricholoma terreum (saponaceolide B7 and saponaceolide M13) (Matsuura et al., 2009; Yin et al., 2014). A study by Yin et al., 2014 also notes that myotoxic saponaceolides have not been identified in T. equestre but states that an extract obtained from this mushroom displayed toxicity of a distinctively different mechanism of action. However, unexpectedly, the description of the methodology and presentation of the results of this study essentially delivers no information on the collected T. equestre specimens, their identification, extract preparation, analytical data nor experimental studies allegedly performed to assess their toxicity (Yin et al., 2014). Due to the publicity that this research has generated, information that T. equestre supposedly contain some toxin has been spread, potentially adding to the conviction that this mushroom should be considered as poisonous. Although T. terreum and T. equestre have their own distinguishable morphological features (for example, T. terreum has a greyish cap and whitish stipe), they are associated with a similar (coniferous) habitat, share a similar fruiting period (late summer‐late autumn) and their geographical distribution in Europe overlaps, thus there is a possibility that less experienced, amateur mushroom foragers can easily be misled. On the other hand, T. terreum has been traditionally considered as an edible mushroom in Europe with fresh specimens collected from the wild being available on the market, and till now no single case of poisoning with this species has been documented. Some investigators challenge the findings by Yin et al. (2014) and argue that T. terreum should remain listed among edible mushroom species by indicating that its content of toxic saponaceolides is ambiguously too low to exert any adverse effects on humans even without considering a potential compound loss during mushroom cooking (Davoli, Floriani, Assisi, Kob, & Sitta, 2016). Additionally, there is no single case of human poisoning linked to T. terreum. One should also note that cases of rhabdomyolysis in humans have also been reported following the consumption of cultivated white button mushroom species Agaricus bisporus whose edibility is well‐established (Akilli, Dündar, Köylü, Günaydın, & Cander, 2014) as well as species from the Boletus and Leccinum genera (Chwaluk, 2013). However, instead of questioning the general safety of these mushrooms, authors have fairly suggested that individual sensitivity could play a role in the development of such symptoms, and that rhabdomyolysis may represent an unspecified reaction, unrelated to specific mushroom species. In summary, the available clinical data on T. equestre toxicity in humans, particularly on its ability to induce rhabdomyolysis, lacks essential information that would enable a clear decision to be made as to whether this mushroom can be the unambiguous cause. No molecular analyses on spores (for example, concentrated from gastric content) or uneaten fruiting bodies were ever performed to deliver precise information on the phylogenetic position of mushrooms involved in poisoning. The reported cases deliver no information on the habitat from which the mushrooms were collected. Such information would be valuable because, as noted in the “Morphological and molecular identification” section, specimens previously identified as T. equestre var. populinum, associated with deciduous trees, are representatives of the T. frondosae clade not the T. equestre group (which is associated with coniferous habitats). Moreover, T. equestre has a long tradition of collection in various geographical regions where it is consumed every year while the overall number of reported poisonings remains low. Therefore, it is possible that some confounding factors (for example, mistaking a mushroom with other, morphologically similar species, inappropriate mushroom storage, individual vulnerability) may be involved in the onset of the described symptoms.

Guidelines for Reporting Poisoning with T. equestre as the Suspected Cause T. equestre poisoning documented so far in scientific literature and the number of gaps or unclear information given in their description, we propose the following guidelines to be considered in any future description of poisonings in which T. equestre is suspected as a causative agent: (i) Provide as much evidence for T. equestre being a causative agent as possible. Claims made by the intoxicated individual that no other mushrooms but T. equestre were consumed should be treated as the least reliable source of information (if any). To confirm that T. equestre may be involved, spore identification in gastric fluids should be performed, although one should note that it may be extremely difficult to distinguish spores of T. equestre relatives (other species than Tricholoma genus). A definitive conclusion can only be drawn from commissioning a molecular analysis on a concentrated spore sample, employing specific mushroom genetic markers ITS and the mitochondrial cox1 gene (which is currently not a routine practice in clinical toxicology). As shown, PCR‐based analyses can be successfully applied for rapid identification of poisonous mushrooms (including those from Tricholoma genus) in gastric content (Kowalczyk et al., 2015 2017

Provide as much evidence for being a causative agent as possible. Claims made by the intoxicated individual that no other mushrooms but were consumed should be treated as the least reliable source of information (if any). To confirm that may be involved, spore identification in gastric fluids should be performed, although one should note that it may be extremely difficult to distinguish spores of relatives (other species than genus). A definitive conclusion can only be drawn from commissioning a molecular analysis on a concentrated spore sample, employing specific mushroom genetic markers ITS and the mitochondrial cox1 gene (which is currently not a routine practice in clinical toxicology). As shown, PCR‐based analyses can be successfully applied for rapid identification of poisonous mushrooms (including those from genus) in gastric content (Kowalczyk et al., (ii) However, one should note that if a patient is being admitted to hospital a few days after mushroom consumption, no spores will be found in gastric content. In some cases, uneaten (for example, frozen) fruiting bodies may still be available directly to relatives of the poisoned person, and would be excellent material for molecular analyses. If no possibility for such confirmation is available, appropriate care should be taken when describing an alleged T. equestre poisoning.

However, one should note that if a patient is being admitted to hospital a few days after mushroom consumption, no spores will be found in gastric content. In some cases, uneaten (for example, frozen) fruiting bodies may still be available directly to relatives of the poisoned person, and would be excellent material for molecular analyses. If no possibility for such confirmation is available, appropriate care should be taken when describing an alleged poisoning. (iii) Report whether the intoxicated individual consumed mushrooms alone or with other subjects, and whether these subjects were also affected. Mushrooms are often consumed as a family meal. A lack of adverse effects observed in meal companions indicates that intoxication is caused by individual susceptibility rather than toxicity of mushroom per se or that part of the meal could consist of poisonous mushrooms erroneously identified as T. equestre .

Report whether the intoxicated individual consumed mushrooms alone or with other subjects, and whether these subjects were also affected. Mushrooms are often consumed as a family meal. A lack of adverse effects observed in meal companions indicates that intoxication is caused by individual susceptibility rather than toxicity of mushroom per se or that part of the meal could consist of poisonous mushrooms erroneously identified as . (iv) Acknowledge the previous history and experience of mushroom consumption. It is important for interpretation whether the poisoned subject had already consumed T. equestre in the past without adverse effects or whether the symptoms occurred after his first lifetime ingestion of the species (or series of consecutive ingestions). The latter scenario may imply individual sensitivity, an indication for in‐depth analysis of its potential basis.

Acknowledge the previous history and experience of mushroom consumption. It is important for interpretation whether the poisoned subject had already consumed in the past without adverse effects or whether the symptoms occurred after his first lifetime ingestion of the species (or series of consecutive ingestions). The latter scenario may imply individual sensitivity, an indication for in‐depth analysis of its potential basis. (v) Acknowledge all possible circumstances associated with T. equestre collection: place of collection (to assess whether significant anthropogenic contamination can be expected) and habitat (coniferous or deciduous), time from transportation to consumption, storage conditions, particularly if consumption had taken place over some number of consecutive days.

Acknowledge all possible circumstances associated with collection: place of collection (to assess whether significant anthropogenic contamination can be expected) and habitat (coniferous or deciduous), time from transportation to consumption, storage conditions, particularly if consumption had taken place over some number of consecutive days. (vi) Acknowledge all possible circumstances associated with T. equestre consumption such as dose at which it was consumed (reported as an amount of fresh mushrooms), form of consumption (for example, fried, boiled, as a soup, and so on), whether the mushroom meal was re‐heated (if yes, the conditions during the storage interval) and whether the mushroom load was consumed along with other food and/or alcohol.

Acknowledge all possible circumstances associated with consumption such as dose at which it was consumed (reported as an amount of fresh mushrooms), form of consumption (for example, fried, boiled, as a soup, and so on), whether the mushroom meal was re‐heated (if yes, the conditions during the storage interval) and whether the mushroom load was consumed along with other food and/or alcohol. (vii) Rule out or clearly acknowledge the presence of all individual factors potentially predisposing to rhabdomyolysis, including: a. alcoholism b. use of narcotics (particularly cocaine, heroin, amphetamines, methamphetamine, phencyclidine, and lysergic acid diethylamide) c. use of pharmaceuticals (particularly statins, fibrates, antihistamines, antibiotics, and psychiatric agents) d. electrolyte imbalance (for example, hypocalcemia, hyponatremia, hypernatremia, hypokalkemia, hypophosphatemia, and nonketotic hyperosmotic state) e. bacterial infections (particularly with Salmonella , Streptococcus pyogenes , Staphylococcus aureus , and Clostridium ), f. viral infections (particularly by coxsackie virus, Epstein‐Barr virus, influenza A or B virus, herpes virus, and primary HIV) g. endocrine disorders (diabetic ketoacidosis, hyperaldosteronism, and hypothyroidism) h. autoimmune disorders (particularly dermatomyositis and polymyositis) i. genetic defects (particularly associated with lipid metabolism, glycolysis, and glycogenolysis)

Rule out or clearly acknowledge the presence of all individual factors potentially predisposing to rhabdomyolysis, including: (viii) Intensity of physical activity before and after mushroom consumption should be acknowledged as in severe form it is known to increase creatine kinase activity, even for a period of few days (Knochel, 1990

Intensity of physical activity before and after mushroom consumption should be acknowledged as in severe form it is known to increase creatine kinase activity, even for a period of few days (Knochel, (ix) In addition to total creatine kinase, report levels of its isoforms, particularly MB isoenzyme which can be an indicator of cardiotoxicity (though use of other cardiac markers such as troponin level is also advised).

In addition to total creatine kinase, report levels of its isoforms, particularly MB isoenzyme which can be an indicator of cardiotoxicity (though use of other cardiac markers such as troponin level is also advised). (x) Provide detailed information on treatment applied before full recovery was achieved. Considering the cases ofpoisoning documented so far in scientific literature and the number of gaps or unclear information given in their description, we propose the following guidelines to be considered in any future description of poisonings in whichis suspected as a causative agent:

Prospects for Future Research Due to the number of gaps with respect to mushroom toxicity, we would like to draw attention to the urgent need for further polyphasic investigations in this field. Firstly, there is still a need to continue molecular analyses of specimens of mushrooms morphologically resembling T. equestre, which are found in different geographical regions and habitats. A species‐wide analytical screening for the presence of myotoxin already identified in mushrooms (for example, cycloprop‐2‐ene carboxylic acid, selected saponaceolides) should be conducted for the Tricholoma genus but also for other genera whose representatives can be mistaken for T. equestre (as outlined in the “Similar species” section). Particularly, the content of saponaceolides, which has only been studied in T. terreum (Feng et al., 2015; Yin et al., 2014), should be further explored. Such analyses could be supported by toxicity assessment using an experimental setting. An interesting in vitro model that could be employed for such a purpose is the use of human skeletal muscle cells isolated from the skeletal muscle of limbs of healthy adults. These cells can now be commercially purchased from certified suppliers and cultured for at least 15 doublings. Such a model allows an assessment of the potential effect of mushroom extracts on the cellular ultrastructural morphology of cells and creatine kinase activities, and appears to be relevant in identification of compound potency to induce rhabdomyolysis in humans. Similar models have already been successfully employed to reproduce myotoxicity induced by statins (Sakamoto & Kimura, 2013; Skottheim, Gedde‐Dahl, Hejazifar, Hoel, & Asberg, 2008). Moreover, it would also be beneficial to study how storage conditions (room temperature, freezing/thawing) and mushroom processing (for example, boiling, stewing, frying, microwaving with water) can affect the chemical quality and microbial composition (particularly occurrence of mold species) of T. equestre fruiting bodies, and exerted toxicity (if any). Considering that T. equestre is still collected and consumed in different countries, a survey on the potential occurrence of toxic effects observed in groups of mushroom foragers would provide a rough estimate on the frequency of adverse effects following its ingestion. There is also a need for detailed molecular analyses within the T. equestre species to establish the magnitude of intraspecific variation and its potential effect on mushroom quality. Last but not least, an effort should be made to evaluate the existence of genetic traits associated with individual susceptibility to T. equestre ingestion. Considering that edible mushrooms representing genera other than Tricholoma have also been reported to induce rhabdomyolysis in humans, the possibility that rare mushroom intolerance can exist in the human population should also be taken into account. If this was the case, a development of a reliable screening method would be urgently needed to identify and protect susceptible subjects.

Conclusion T. equestre cannot be considered toxic per se and does not appear to exhibit any greater health threat than other mushroom species which are currently considered as edible. Based on reported element levels in fruiting bodies, an expected intake of metal and metalloids following a consumption of 300 g of fresh mushrooms should not pose a risk. No toxic compounds, including causative agents of rhabdomyolysis, have ever been identified in T. equestre. Considering the available in vivo and clinical data on myotoxicity of various mushrooms, and clues from molecular analyses of Tricholoma, 3 hypotheses can be put forward: All (or most) edible mushrooms can induce rhabdomyolysis in humans at high and repeated doses. Rhabdomyolysis induced by edible mushrooms is a reaction related to as yet unidentified genetic traits. Rhabdomyolysis has been triggered by the consumption of morphologically related but genetically distinctive mushroom species to T. equestre. Based on available evidence we are of the opinion thatcannot be considered toxic per se and does not appear to exhibit any greater health threat than other mushroom species which are currently considered as edible. Based on reported element levels in fruiting bodies, an expected intake of metal and metalloids following a consumption of 300 g of fresh mushrooms should not pose a risk. No toxic compounds, including causative agents of rhabdomyolysis, have ever been identified in. Considering the availableand clinical data on myotoxicity of various mushrooms, and clues from molecular analyses of, 3 hypotheses can be put forward: Further research to evaluate potential myotoxic compounds in morphologically similar mushroom species to T. equestre is urgently required. All future studies on T. equestre should establish a phylogenetic position of tested specimens using available molecular tools. Particular care should be taken when reporting cases of poisoning with T. equestre to avoid hype and mis‐ or overinterpretation of data.

Conflict of Interest None to declare.

Authors Contribution Piotr Rzymski and Piotr Klimaszyk contributed equally to this manuscript by providing ideas, researching studies, and writing the manuscript.