Arsenic in Mushrooms - Hyperaccumulation and Speciation
Dissertation
Zur Erlangung des akademischen Grades einer Doktorin an der naturwissenschaftlichen Fakultät der Karl-Franzens-Universität Graz
Vorgelegt von
Simone Braeuer, MSc
Institut für Chemie, Analytical Chemistry for Health and Environment
Juni 2018 The work for this thesis was carried out at the Institute of Chemistry, in the research group Analytical Chemistry for Health and Environment, under the supervision of Ao. Univ. Prof. Dr. Mag. Walter Goessler from October 2014 to June 2018.
Simone Braeuer
June 2018 Danksagung
An erster Stelle möchte ich meinem Betreuer Walter Goessler danken. Danke für die intensive Betreuung, die vielfältigen Themen, die Reisen zu Konferenzen und Projektpartnern, die Denkanstöße und den Freiraum für meine eigenen Ideen. Danke auch dafür, dass ich so ein spannendes Thema wiederaufgreifen durfte, über 20 Jahre nach deinen ersten Erfahrungen damit.
Lieber Matthias: Danke, dass du mich aushältst und mir immer wieder vor Augen führst, dass das Leben auch zum Genießen da ist. Es ist eigentlich verrückt und unverantwortlich, wenn Lebenspartner gleichzeitig an ihrer Dissertation arbeiten. Danke, dass wir es durch alle stressigen und nervenraubenden Situationen, durch alle Höhen und Tiefen, bis hierher geschafft haben.
Ganz besonders danke ich auch meinen Eltern. Liebe Mama, lieber Papa, ich danke euch für eure Unterstützung und euer Verständnis in jeder Hinsicht und zu jeder Zeit, für euren Rat, die Einladungen zum Sonntagsessen, die Urlaube, das Schifahren, die Fahrradreparaturen und das Saunieren. Danke, dass ich bei euch immer ein offenes Ohr für all meine großen und kleinen Probleme finde. Ich kann mir keine besseren Eltern vorstellen.
Natürlich danke ich auch meinen Geschwistern Susi, Florian und Stefan. Danke, dass ihr mich immer wieder auf andere Gedanken bringt, mit und ohne Copper. Ich bin stolz, eure große Schwester zu sein.
Vielen Dank allen MitarbeiterInnen der Analytischen Chemie, und besonders der ACHE Arbeitsgruppe. Obwohl wir so unterschiedliche Charaktere sind, sind wir doch ein gut ausgeglichener Haufen.
Danke an Lisa und Kathi (und Melanie und Karin). Im Labor und im Büro seid ihr immer für mich da und hört euch geduldig an, was mich beschäftigt. Ihr helft mir, Probleme von einem anderen Blickwinkel zu sehen. Danke, dass ich auf euch zählen kann, wenn bei mir wieder einmal alles drunter und drüber geht.
Den Herren in der Runde, Oliver, Chris und Stefan (und Franz), danke ich von Herzen für die geduldige Unterstützung in technischen Herausforderungen.
Ein großer Dank gilt Jaqui, Katharina und Antonia. Auf euc h kann ich mich immer verlassen, und das weiß ich wirklich sehr zu schätzen.
I also want to thank Jan Borovička, who not only provided me with many of the samples for my experiments and nice pictures for the thesis, but also initiated the joint project.
Thank you, Kenneth Jensen, for the measurements with the Orbitrap and the fast results.
Vielen Dank auch an Toma Glasnov und Gema Guedes de la Cruz für die unkomplizierte Synthese des AC2. Des Weiteren bedanke ich mich beim Arbeitskreis Heimische Pilze vom Universalmuseum Joanneum, allen voran Gernot Friebes, Uwe Kozina und Seppi Flack für das eifrige Sammeln und Bereitstellen von steirischen Pilzen. Michaela und Gernot Friebes danke ich außerdem für die schönen Pilzfotos.
Danke an alle StudentInnen und FerialpraktikantInnen für die Unterstützung im Labor: Elisa Prall, Daniel Vetter, Sarah Schlagenhaufen, Sophia Schlagenhaufen, Herbert Becker, Martin Walenta und Lukas Rodziewicz. Danke nicht nur für die Arbeit, die ihr mir abgenommen habt, sondern auch die Gelegenheit, mich durch euch didaktisch weiterzuentwickeln.
Schließ lich danke ich dem FWF für die Finanzierung meiner Doktorarbeit im Rahmen des Projektes I2352-B21.
Danke ! Table of Contents
Abstract ...... 1
Zusammenfassung...... 2
Publications and presentations...... 3
1 Introduction...... 6
1.1 Fungi ...... 6
1.2 Element accumulation...... 7
1.2.1 General aspects ...... 7
1.2.2 Element accumulation by macrofungi...... 9
1.2.3 Arsenic accumulation by macrofungi...... 11
1.3 Arsenic species ...... 12
1.3.1 Introduction...... 12
1.3.2 Arsenic and its species in the environment...... 14
1.3.3 Arsenic species in macrofungi ...... 18
1.3.4 Arsenic biotransformation in macrofungi? ...... 23
1.4 Toxicity and regulations on arsenic...... 26
1.5 Techniques for arsenic speciation analysis in environmental samples...... 27
2 Overview of the publications ...... 34
3 Experimental ...... 40
4 Publications ...... 48
4.1 Publication 1: Arsenic hyperaccumulatio n and speciation in the edible ink stain bolete (Cyanoboletus pulverulentus )...... 48
4.2 Publication 2: A unique arsenic speciation profile in Elaphomyces spp. ("deer truffles") - trimethylarsine oxide and methylarsonous acid as significant arsenic compounds...... 63
4.3 Publication 3: Homoarsenocholine - a novel arsenic compound detected for the first time in nature ...... 80
5 Conclusion and outlook...... 92
6 References...... 95 Abstract
Abstract
Fungi are important organisms in our environment and unique in many ways. For example, some macrofungi are able to accumulate remarkable concentrations of (trace) elements in their fruit-bodies. One of these elements is arsenic, where up to 7000 mg/kg dry mass (dm) have been found in the crown cup ( Sarcosphaera coronaria ). Such concentrations are hardly ever reported for any natural living organism. Amongst macrofungi, the ability to accumulate arsenic is species specific, but the soil is also regarded as influencing factor. Within this thesis, a new hyperaccumulating mushroom was identified, with up to 1300 mg As/kg dm: The edible ink stain bolete Cyanoboletus pulverulentus . Because the arsenic was present as the probably carcinogenic dimethylarsinic acid, frequent consumption of this mushroom should be avoided.
Mushrooms not always contain just one arsenic species, which was demonstrated with samples of the genera Elaphomyces (deer truffles) and Ramaria (coral mushrooms). Elaphomyces samples contained methylarsonic acid as main arsenical, but also high concentrations of trimethylarsine oxide. Both compounds are only detected in trace concentrations or even not at all in most other macrofungi. Strikingly, there were also significant concentrations of the otherwise rarely detected methylarsonous acid.
The major arsenic compound in samples of the genus Ramaria was arsenobetaine. Although otherwise only found in the marine environment, this compound is often reported as the main arsenical in mushrooms. More remarkable was therefore the detection of dimethylarsinoylacetate and trimethylarsoniopropionate. These compounds are sometimes found in the marine biota, but have never been described for terrestrial samples before. Finally, the arsenic species (3-hydroxypropyl) trimethylarsonium ion that we named homoarsenocholine was identified for the first time ever in nature and could be an important compound for the elucidation of the biotransformation pathways of arsenic.
1 Zusammenfassung
Zusammenfassung
Pilze sind wichtige Bestandteile unserer Umwelt und in vielerlei Hinsicht einzigartig. So können sie zum Beispiel extreme Konzentrationen von (Spuren-)Elementen anreichern. Im Fall von Arsen wurden bereits bis zu 7000 mg /kg Trockenmasse (dm) im Kronenbecherling ( Sarcosphaera coronaria) gefunden. Kaum ein anderer Organismus enthält solch hohe Arsenkonzentrationen. Die Fähigkeit Arsen anzureichern hängt von der Pilzspezies ab, aber auch die Erde unter den Pilzen gilt als Einflussfaktor. In dieser Dissertation wurde eine neue hyperakkumulierende Pilzspezies entdeckt, mit bis zu 1300 mg As/kg dm, der schwarzblauende Röhrling Cyanoboletus pulverulentus . Da das Arsen als vermutlich krebserregende Dimethylarsinsäure vorlag, sollte ein regelmäßiger Verzehr dieses Pilzes vermieden werden.
Die Arsenspeziation von Pilzen besteht nicht immer nur aus einer Verbindung, was anhand der Gattungen Elaphomyces (Hirschtrüffel) und Ramaria (Korallenpilze) gezeigt wurde. Hirschtrüffel enthielten Methylarsonsäure als Hauptspezies, aber auch hohe Konzentrationen an Trimethylarsinoxid. Beide Spezies kommen in den meisten anderen Pilzen höchstens in Spuren vor. Erstaunlicherweise wurden auch signifikante Konzentrationen der sehr selten vorkommenden dreiwertigen methylarsonigen Säure gefunden.
Die dominierende Arsenverbindung in Ramaria Proben war Arsenobetain. Diese Spezies wird zwar normalerweise der marinen Umwelt zugeschrieben, ist aber auch häufig die Hauptspezies in Pilzen. Außergewöhnlicher war daher die Entdeckung von Dimethylarsinoylacetat und Trimethylarsinopropionat. Diese Spezies sind zwar hin und wieder in marinen Organismen präsent, wurden bis jetzt allerdings nicht in terrestrischen Proben detektiert. Außerdem konnte eine vollkommen neue natürliche Arsenverbindung identifiziert werden das (3-hydroxypropyl) trimethylarsonium Ion, welche Homoarsenocholin genannt wurde. Diese könnte einen wichtigen Baustein für die Aufklärung der Biotransformierungsmechanismen von Arsen darstellen.
2 Publications and presentations
Publications and presentations
Publications this thesis is based on
Braeuer S, Borovička J, Glasnov T, La Guedes de Cruz G, Jensen KB, Goessler W ( 2018 ) Homoarsenocholine – A novel arsenic compound detected for the first time in nature. Talanta 188:107 –110
Braeuer S, Borovička J, Goessler W ( 2018 ) A unique arsenic speciation profile in Elaphomyces spp. (“deer truffles”)— trimethylarsine oxide and methylarsonous acid as significant arsenic compounds. Anal. Bioanal. Chem. 410:2283 –2290
Braeuer S, Goessler W, Kameník J, Konvalinková T, Žigová A, Borovička J ( 2018 ) Arsenic hyperaccumulation and speciation in the edible ink stain bolete ( Cyanoboletus pulverulentus ). Food Chem. 242:225 –231
Other publications
Kaňa A, Koplík R, Braeuer S, Goessler W, Mestek O ( 2018 ) Analysis of Main Arsenic Species in Canned Fish Marketed in the Czech Republic and Austria. J. Food Chem. Nanotechol. 4(1):10 –17
Braeuer S, Dungl E, Hoffmann W, Li D, Wang C, Zhang H, Goessler W ( 2017 ) Unusual arsenic metabolism in Giant Pandas. Chemosphere 189(Supplement C):418 –425
Borovička J, Braeuer S, Žigová A, Gryndler M, Dima B, Goessler W, Frøslev TG, Kameník J, Kärcher R (2017) Resurrection of Cortinarius coalescens . Taxonomy, chemistry, and ecology. Mycol. Prog. 66:250
Myrissa A*, Braeuer S*, Martinelli E, Willumeit-Römer R, Goessler W, Weinberg AM ( 2017 ) Gadolinium accumulation in organs of Sprague-Dawley® rats after implantation of a biodegradable magnesium-gadolinium alloy. Acta Biomater. 48:521 –529 *These authors contributed equally to the publication
Neamtiu I, Bloom MS, Gati G, Goessler W, Surdu S, Pop C, Braeuer S, Fitzgerald EF, Baciu C, Lupsa IR, Anastasiu D, Gurzau E ( 2015 ) Pregnant women in Timis County, Romania are exposed primarily to low-level (10 μg/l) arsenic through residential drinking water consumption. Int. J. Hyg. Environ. Health 218(4):371 –379
Conference proceedings
Braeuer S, Goessler W ( 2014 ) Arsenic and its compounds in tissue samples from Austrian cattle. In: Litter MI, Nicolli HB, Meichtry JM, Quici N, Bundschuh J, Bhattacharya P, Naidu R (eds) One Century of the Discovery of Arsenicosis in Latin America (1914-2014). As 2014. Taylor & Francis Group , London, pp 438 –439
3 Publications and presentations
Goessler W, Braeuer S ( 2014 ) Unusual arsenic speciation in urine of ruminants. In: Litter MI, Nicolli HB, Meichtry JM, Quici N, Bundschuh J, Bhattacharya P, Naidu R (eds) One Century of the Discovery of Arsenicosis in Latin America (1914-2014). As 2014. Taylor & Francis Group , London, pp 427 – 430
Pérez Carrera A, Alvarez Gonçalvez C, Fernández Cirelli A, Braeuer S, Goessler W ( 2014 ) Arsenic levels in bovine kidney and liver from an arsenic affected area in Argentina. In: Litter MI, Nicolli HB, Meichtry JM, Quici N, Bundschuh J, Bhattacharya P, Naidu R (eds) One Century of the Discovery of Arsenicosis in Latin America (1914-2014). As 2014. Taylor & Francis Group , London, pp 436 – 437
Oral presentations Braeuer S, Goessler W. ( 2018 ) Arsenic accumulation and speciation in mushrooms. YISAC , University of Graz, Graz, Austria.
Braeuer S, Borovička J, Glasnov T, La Guedes de Cruz G, Jensen KB, Goessler W (2018 ) Homoarsenocholine in mushrooms . International Conference on Analytical Sciences and Spectroscopy , Toronto, Canada.
Braeuer S, Borovička , J, Goessler W ( 2017 ). Exploring the arsenic speciation in mushrooms. ASAC JunganalytikerInnen Forum, BOKU Wien, Vienna, Austria.
Braeuer S ( 2017 ) Investigating environmental samples with (HPLC-) ICPQQQMS. European Winter Conference on Plasma Spectrochemistry, Agilent Technologies Session , St. Anton am Arlberg, Austria. - Invited presentation
Braeuer S, Borovička , J, Goessler W ( 2017 ). Not only seafood can contain a lot of arsenic. European Winter Conference on Plasma Spectrochemistry , St. Anton am Arlberg, Austria.
Braeuer S ( 2016 ) Pilze und Arsen - eine heimliche Liebe? (LC-ICP-MS Speziationsanalytik). Agilent Technologies Anwendertreffen für Elementspektroskopie, Graz, Austria. - Invited presentation
Braeuer S, Hoffmann W, Dungl E, Tang C, Li D, Goessler W. ( 2016 ) Arsenspeziation im Urin von Großen Pandas. ICPMS Anwendertreffen , University of Siegen, Siegen, Germany.
Braeuer S ( 2016 ) Internationale Arsenküche. Science Slam , Graz, Austria.
Braeuer S, Borovička, J, Goessler W ( 2016 ) Arsenic speciation in different mushroom families. Doc Days , University of Graz, Graz, Austria.
Braeuer S, Čadková Z, Száková J, Tlustoš P, Goessler W. ( 2015 ) Are wild small mammals suited as bioindicators for trace element levels in the environment? Doc Days , TU Graz, Graz, Austria.
4 Publications and presentations
Braeuer S, Čadková Z, Száková J, Tlustoš P, Goessler W. ( 2015 ) Mouse ≠ mouse, or: Wild small terrestrial mammals as possible bioindicators for trace element levels in the environment. European Winter Conference on Plasma Spectrochemistry, University of Münster, Münster, Germany.
Braeuer S, Alvarez Gonçalvez C, Pérez-Carrera A, Fernández Cirelli A, , Goessler W ( 2014 ) Arsen und seine Spezies in Milch- und Gewebeproben Österreichischer und Argentinischer Rinder. ICPMS Anwendertreffen, Helmholtz-Zentrum Geesthacht, Geesthacht, Germany.
Braeuer S, Pérez-Carrera A, Fernández Cirelli A, Alvarez Gonçalvez C, Goessler W ( 2014 ) Comparison of the elemental composition of Austrian and Argentinean bovine tissue samples. BNASS – TraceSpec Tandem Conference, University of Aberdeen, Aberdeen, United Kingdom.
Braeuer S, Goessler W ( 2013 ) Arsenic Speciation in Tissue Samples of Cattle from Argentina Exposed to Arsenic via Groundwater. ASAC JunganalytikerInnen Forum , TU Wien, Wien, Austria.
Poster presentations Braeuer S, Goessler W, Milić Mi, Šerić V, Milić Ma, Pavičić I, Marjanović Čermak AM, Bonassi S, Oreščanin V, Vinković Vrček I. ( 2018 ) Can measurement of arsenic concentration and other elements be comparable in human buccal cells, hair samples and urine samples and correlate with DNA damage assessment with micronucleus buccal cytome assay? Congress of Toxicology in Developing Countries , Belgrade, Serbia.
Braeuer S, Borovička, J, Goessler W ( 2017 ) Rekordverdächtige Arsenkonzentrationen in Pilzen. ANAKON , University of Tübingen, Tübingen, Germany. Winner of a poster prize.
Braeuer S, Goessler W ( 2015 ) Determination of the chemical forms of arsenic in beef tissues. International Student Congress , Medical University of Graz, Graz, Austria. Winner of a poster prize.
Braeuer S, Goessler W ( 2014 ) Arsenic and its Compounds in Tissue Samples from Austrian Cattle. Fifth International Congress on Arsenic in the Environment (As2014) , Buenos Aires, Argentina. – Winner of a poster prize.
5 Introduction
1 Introduction
1.1 Fungi Fungi constitute the kingdom of Fungi, which is separated from plants and animals. So-called macrofungi are fungal species that belong to the divisions of Basidiomycota or Ascomycota. During their reproductive live stage, they are able to form fruit-bodies (sporocarps), which are commonly called mushrooms [1]. In the vegetative live stage, only the mycelium is present. Most of the typical mushrooms are basidiomycetes and have a similar morphology, depicted in Figure 1. Representatives of the division of Ascomycota are for example yeast, morels and truffles. Truffles are so-called hypogeous fungi, as their fruit-bodies are growing underground, opposed to epigeous fungi, growing aboveground [2].
Figure 1. Morphology of typical basidiomycetes with the common terms for the different parts. © Simone Braeuer. *Tubes or gills: depends on fungal species; both are the spore-forming part (the hymenium) of the mushroom. **Ring: Presence depends on fungal species.
The average lifetime of sporocarps is around 10 to 14 days, and their dry mass (dm) is usually between 6 and 14 %[3]. Commonly, 10 % is used for calculations, and one meal is calculated as 300 g of fresh mushroom [4].
Mushrooms are often valued for their high protein content, which is between 10 and 30 % dm [5, 6]. There are also high concentrations of carbohydrates, for example structural polysaccharides like chitin, but the lipid content is usually only a few % dm [5]. The energy of fresh mushrooms is 350 – 400 kcal/kg
[6]. Sporocarps can also contain several vitamins, including vitamins B 1, B 2, B 3, C, D and E [5]. Volatile compounds, especially 1-octen-3-ol, make up the typical mushroom flavor, while free amino acids and 5’ -nucleotides are responsible for the taste [7].
6 Introduction
Fungi are not only interesting as food; they also play important roles in the environment. For example, saprotrophic (or: saprobic) fungi feed on dead organisms and hence are vital of the decay of organic matter. Further, mycorrhizal fungi live in symbiosis with plants (e.g. trees). The fungal mycelium and the plant’s roots form together the mycorrhizae (see Figure 2), which enable the exchange of nutrients between fungus and plant. It can be distinguished between endomycorrhizal and ectomycorrhizal fungi, where only the first ones penetrate the cell walls of the plant. In both types, the fungus can regulate the uptake and transfer of nutrients and toxic substances to its host plant ( “homeostasis ”). The different regulation mechanisms are described in detail by others [8, 9]. Besides the storage in the fungal vacuoles or binding of the toxic elements, e.g. by metallothioneines, another possible way of protecting the host plant from high element concentrations is by transporting the elements to the sporocarps. Indeed, fruit-bodies can accumulate immense concentrations of certain elements, often not representative of neither the total element concentration nor its bioavailable part in the underlying soil [8].
Figure 2. Cleaned ectomycorrhizae of Imleria badia. © Jan Borovička . 1.2 Element accumulation
1.2.1 General aspects The element concentrations in sporocarps are influenced primarily by the mushroom species, the elements’ soil concentrations and their chemical forms in the soil (or, more generally, substrate), and to a lesser extent by the age of the mycelium and the age and size of the fruit-body [4]. Many fungal species take up certain elements very selectively, which can even be different for elements with similar chemical properties [10]. In some cases, the element accumulation is so species- or genus-specific that the use of element concentrations for taxonomic characterization of macrofungi was suggested [11]. In most cases, the elements are not evenly distributed within the fungal fruit-body. There is no generally valid rule, but the highest element concentrations are often found in the spore-forming part of the mushroom, such as gills or tubes, whereas the stipe usually contains lower concentrations [12, 13]. It has to be pointed out that fruit-bodies are prone to contamination by soil, especially concerning
7 Introduction major soil elements such as iron, calcium or aluminum, and careful washing is essential to reduce a possible contamination by soil particles to a minimum [14]. Because of its close proximity to the ground, the stipe is probably the most affected part of the mushroom.
Some fungal species are behaving as so-called excluders, which means that they avoid the uptake of certain elements from the substrate, leading to very low concentrations in these organisms. However, when the soil concentration of these elements is too high, the sporocarp loses this function and starts to take up excessively high amounts of the element [15].
On the other hand, as already mentioned, there are macrofungi that are able to accumulate certain elements, depending on the fungal species. Fungi that can accumulate exceptionally high concentrations of an element are often referred to as hyperaccumulators. This term was first used in 1977 for plants that contained more than 1000 mg/kg dm of nickel [16]. Since then, hyperaccumulating plants have been identified for several elements [17]. The first living organism that was found to hyperaccumulate arsenic (> 1000 mg/kg dm) was the fern Pteris vittata in 2001 [18]. Van der Ent et al. reviewed the correct use of the term “hyperaccumulation ” and the hyperaccumulation threshold criteria in plants [19]. They suggested updated individual limit concentrations for several elements (Table 1), depending mainly on their typical concentrations in non-accumulators and in soil. They point out that the bioconcentration factor alone (the ratio of the concentrations in shoot and soil) is a poor indication of hyperaccumulation [19]. Sometimes, hyperaccumulators are simply defined as species where the concentrations of the specific element are at least 100 times higher than in other species that grow under similar conditions [20].The hyperaccumulation limits for plants are usually also applied for mushrooms, since there are no separate definitions for macrofungi. One exception is silver, where Borovička et al. proposed separate threshold limits for ectomycorrhizal and saprotrophic macrofungi [21]. The reason why plants and fungi (hyper) accumulate elements is not yet understood. One hypothesis is that elements are accumulated as a defense mechanism against enemies [22]. High element concentrations in fruit-bodies of mycorrhizal fungi could also be a protection mechanism, as already mentioned before: To save the plant from toxic concentrations of an element, the fungus stores it in its sporocarps [8].
Table 1. Threshold criteria for hyperaccumulation of elements in plants, suggested by [19].*Ag value for macrofungi taken from [21]. Elements Threshold limit [mg/kg dm] Cd, Se, Tl 100 Ag* 100 (ectomycorrhizal fungi), 300 (saprotrophic fungi) Co, Cr, Cu 300 As , Ni, Pb 1000 Zn 3000 Mn 10000
8 Introduction
1.2.2 Element accumulation by macrofungi Hyperaccumulation in macrofungi has only been detected for arsenic, silver and perhaps copper so far. The case of arsenic is discussed in more detail below. Silver is hyperaccumulated by Amanita strobiliformis and A. solitaria , where up to 1200 mg Ag/kg dm have been found [23]. Also up to 1200 mg/kg dm, but this time of copper, have been reported in Thelephora terrestris (In German: Fächerförmiger Erdwarzenpilz, Figure 3A) [24]. It has to be noted that the samples originated from an old mine with elevated soil concentrations of copper and other metals, and that this specific macrofungal species can be very difficult to clean. In comparison, two samples of T. terrestris that were analyzed during this doctoral thesis only contained 12 and 45 mg Cu/kg dm. On the other hand, four samples of Thelephora penicillata (In German: Weißer Warzenpilz, Figure 3B) contained between 490 and 820 mg Cu/kg dm. The two T. terrestris and the four T. penicillata samples were all from uncontaminated areas.
A B
Figure 3. A: Thelephora terrestris, B: Thelephora penicillata. Both pictures: © Jan Borovička.
In contrast to hyperaccumulation, simple accumulation (in other words; high, but not extreme concentrations in comparison to other species) by mushrooms has been reported for many elements. This topic, in combination with typical element concentrations in non-accumulating mushrooms was reviewed a couple of times over the last 20 years by Kalač [3, 4, 6, 13]. A summary of normal element concentrations and probable accumulating macrofungi is given in Table 2.
One of the first identified element accumulators amongst macrofungi was Amanita muscaria (the well- known fly agaric, in German: Fliegenpilz). Already in 1931, ter Meulen found around 30 mg V/kg dm [25], and Bertrand reported up to 180 mg V/kg dm in A. muscaria , while other investigated mushrooms, including other species of the genus Amanita , contained less than 3 mg V/kg dm [26]. Another famous example is Boletus edulis , commonly known as king bolete, cep or porcino (in German: Steinpilz), one of the most often collected and widely consumed wild mushrooms. It can accumulate high concentrations of selenium (median of 345 samples: 35 mg /kg dm), but also up to 14 mg/kg dm of mercury [27] (median of 345 samples: 3.08 mg/kg dm) [28].
9 Introduction
Table 2. Normal element concentrations [mg/kg dm] in non-accumulators and fungal species that have been reported to be accumulators, adapted from [6, 13]. Arsenic is discussed separately in the text. Element Normal ranges Reported as accumulators Aluminium 20 –150 Amanita rubescens, Leccinum scabrum, Xerocomus chrysenteron Antimony < 0.1 Suillus spp ., Laccaria amethystina, Amanita rubescens Barium 2–4 Beryllium < 0.05 –0.5 Boron 5–15 Marasmius wynnei Bromine 1–20 Lepista gilva, L. inversa, Amanita rubescens Cadmium 0.5 –5 Agaricus spp., especially Agaricus macrosporus Caesium 0.5 –10 Suillus luteus Calcium 100 - 500 Morchella spp . Chlorine 1000-6000 Chromium 0.5 –5 Morchella elata, Armillaria mellea, Macrolepiota procera, Marasmius oreades, Agaricus spp ., Lactarius deliciosus Cobalt < 0.5 Ramaria largentii, Hygroporus eburneus, Cantharellus cibarius, Agaricus arvensis Copper 10 –100 Agaricus macrosporus, A. silvicola, M. procera, M. rhacodes Gallium < 0.15 Gold < 0.02 Lycoperdon perlatum, Cantharellus lutescens, Morchella esculenta Iodine 0.07 –0.5 Calvatia excipuliformis, Macrolepiota procera, Lepista nuda, Amanita rubescens, Agaricus spp., Lactarius deliciosus Iron 50 –300 Suillus variegatus, S. luteus, Armillaria mellea, Hygrophoropsis aurantiaca Lead < 1 –5 Macrolepiota rhacodes, M. procera, Lycoperdon perlatum Agaricus spp ., Lepista nuda Lithium 0.1 –0.2 Craterellus cornucopioides, Amanita strobiliformis Magnesium 800 - 1800 Manganese 10 –60 Boletus edulis, Macrolepiota procera, Agaricus spp . Mercury < 0.5 –5 Boletus edulis, B. pinophilus, Calocybe gambosa, Agaricus spp ., Macrolepiota procera, M. rhacodes, Lepista nuda Molybdenum <0.60 Nickel < 1 –15 Laccaria amethystina, Coprinus comatus, Leccinum spp . Phosphorus 5000 – 10000 Potassium 20000 – 40000 Rubidium 10 - 100 Boletaceae family Selenium < 2 –20 Albatrellus pes-caprae, Boletus edulis, B. pinicola, B. aestivalis, I. badia Silver < 10 Amanita strobiliformis, Agaricus spp., M.procera, B.edulis, L.perlatum Sodium 100 - 400 Strontium < 2 Amanita rubescens Sulfur 1000 – 6000 Thallium < 0.3 Tin < 0.5 Titanium < 10 Vanadium < 0.5 Coprinus comatus, toxic Amanita muscaria Zinc 20 –200 Calvatia utriformis, Lycoperdon perlatum, Suillus variegatus, S. luteus Lanthanides < 1 (Ce > La > Nd)
10 Introduction
1.2.3 Arsenic accumulation by macrofungi Most non-accumulating mushrooms contain less than 10 or even less than 1 mg As/kg dm [3, 6, 28 – 32]. Arsenic concentrations are in general higher in caps than in stipes [33, 34], although this cannot be taken as a strict rule, and opposing results (higher concentrations in the stipe or not significant differences at all) can be found in literature as well [35, 36].
The ability to hyperaccumulate arsenic was first detected in the so-called crown cup Sarcosphaera coronaria (in German: Kronenbecherling; Figure 4A) by Stijve et al., who found between 360 and 2100 mg As/kg dm [37]. In another study, 2100 mg As/kg dm were detected in one sample of S. coronaria as well, while a second sample of the same species contained 340 mg As/kg dm [38]. The world record of 7100 mg As/kg dm was reported by Borovička [39]. The second macrofungal species where over 1000 mg As/kg dm have been found is Laccaria amethystina, also known as amethyst deceiver (In German: Violetter Lacktrichterling, Figure 4B), where one sample from a contaminated site contained 1420 mg As/kg dm [40]. Already in 1983, the arsenic concentrations of 12 samples of L. amethystina were determined [41]. They ranged from 34 to 182 mg/kg dm, with a median of 77 mg/kg dm. Further, two samples of L. amethystina that were collected in Slovenia contained 34 and 405 mg As/kg dm [38]. In another study, several samples of L. amethystina from pristine areas were investigated [42]. They also had quite high arsenic concentrations, namely on average 59 ± 55 mg As/kg dm, with a range of 4.1 – 147 mg As/kg dm [42]. The only other sporocarp that has been reported with more than 1000 mg As/kg dm so far is a sample of Lycoperdon pyriforme (pear- shaped puffball, in German: Birnenstäubling, Figure 4C), which was collected in the vicinity of a gold mine in Yellowknife, Canada, and contained 1010 mg As/kg dm [43]. In another study, samples of the genus Lycoperdon from uncontaminated sites contained only up to around 3 mg As/kg dm [44]. Notably, all three hyperaccumulating muchrooms ( S. coronaria , L. amethystina and L. pyriforme, Figure 4) are in general classified as edible, even though they are probably not very well known and hence only collected by few people.
A B C
Figure 4. Arsenic hyperaccumulators. A: Sarcosphaera coronaria, B: Laccaria amethystina, C: Lycoperdon pyriforme. A, B &C: © Jan Borovička .
Other mushroom samples from Yellowknife, investigated in the same study as the already mentioned L. pyriforme, but collected at the site of a second gold mine, contained up to 36 mg As/kg dm ( Paxillus involutus, Psathyrella candolleana, Leccinum scabrum) , and a sample of Coprinus comatus from the
11 Introduction first gold mine contained 410 mg As/kg dm [43]. Individual samples of Sarcodon imbricatum and Entoloma lividum from pristine areas also contained quite high arsenic concentrations, namely 23.8 and 38.9 mg/kg dm, respectively [38]. Another mushroom with elevated arsenic concentrations is Laccaria fraterna, where 30 mg As/kg dm were found [44].
As can be seen, the arsenic concentration is highly dependent on the fungal species. Vetter found large differences in the arsenic concentrations of samples of different fungal species collected at the same site [42]. In a lab experiment, it could be shown that the arsenic uptake under the same conditions can vary a lot between different mushroom species [45]. Nearing et al. argued that the taxonomic position (e.g. family, genus or species) of the mushrooms is an important factor, probably the most important one, that influences the arsenic concentration [46]. In addition, the arsenic concentration of the underlying soil can influence the arsenic concentration in the fungal fruit-bodies, as can already be seen from the examples above. Also, samples of the genus Boletus , collected in China, contained between 0.1 and 120 mg As/kg dm [35]. The highest concentrations were attributed to elevated arsenic concentrations in the soil. A study by Mleczek et al. showed that Imleria badia (also called Boletus badius or commonly just bay bolete, in German: Maronenröhrling ) is heavily susceptible to pollution of the underlying soil [47]. While samples of I. badia from pristine areas contained less than 1 mg As/kg dm, samples from contaminated sites had between 50 and 489 mg As/kg dm. In the same publication the authors showed that the arsenic concentrations were quite constant in mushrooms repeatedly collected at the same site over the course of four years [47].
1.3 Arsenic species
1.3.1 Introduction Arsenic is occurring in our environment in many different chemical forms. The names, abbreviations and structures of the arsenic species that are more or less relevant for this thesis are shown in Table 3. Because the different arsenic species exhibit different toxicities (see chapter 1.4), it is essential to determine not only the total arsenic concentrations, but also the arsenic speciation in foodstuff and drinks. A second huge topic in the field of arsenic research is the biogeochemical cycling of the element. It is still poorly understood where, how and why arsenic species are transformed in the environment. From the 1980s on, a lot of work was carried out to understand the arsenic metabolism of terrestrial mammals [48, 49]. Most of the recent studies have been focusing on the marine biota [50]. Within this environment, special attention has been given to lipid-soluble arsenic species [51].
12 Introduction
Table 3. Known naturally occurring arsenic species relevant for this thesis. pK of TMAO taken from [52], pK of AB and DMAA taken from [53].
Arsenic acid Methylarsonic acid Dimethylarsinic acid Trimethylarsine oxide [arsenate, As(V)], (methylarsonate, MA) (dimethylarsinate, (TMAO), pK 1-3 : 2.2, 6.9, 11.5 pK 1-2 : 4.1, 9.1 DMA), pK: 6.3 pK: 2.7
Arsenous acid Methylarsonous acid Dimethylarsinous acid Trimethylarsine [arsenite, As(III)] [methylarsonite, [Dimethylarsinite, (TMA) pK 1-3 : 9.2, 13.5, 14.0 MA(III)] DMA(III)]
Tetramethyl- Trimethylarsonio- Arsenocholine Arsenobetaine arsonium ion propionic acid (AC) (AB), pK: 2.1 (TETRA) (TMAP, AB2)
Dimethylarsinoylethanol Dimethylarsinoylacetic acid Dimethylarsinoylpropionic acid (DMAE) (DMAA), pK: 4.0 (DMAP)
Lipid soluble arsenic species Dimethylarsinoylbutanoic acid (DMAB) (here: Hydrocarbon AsHC 332 from [54])
Dimethyl-(5-ribosyl)arsine oxide + R Phosphate sugar (arsenosugar)
Glycerol sugar Sulfonate sugar Sulfate sugar
13 Introduction
1.3.2 Arsenic and its species in the environment In uncontaminated waters, typical arsenic concentrations are below 2 µg/L [55]. In natural water, usually only arsenite and arsenate can be found [56], but in very sulfidic waters, thiolated inorganic arsenic species can be present as well [57]. It has been shown lately that the volatile trivalent TMA is formed and released from natural seawater [58]. TMAO, either the oxidation product or reduction substrate of TMA, can be regarded as the methylation product of inorganic arsenic (via MA and DMA), or also as a degradation product of AB, the major arsenical in marine fish.
The total arsenic concentration in uncontaminated soil is around 2-20 mg/kg [59]. When soil samples are extracted with aqueous or methanolic solutions, mainly inorganic arsenic is found in most cases [60]. Sometimes, low concentrations of other arsenicals can also be found, including MA, DMA, TMAO and AB [61].
Typical plant materials from uncontaminated areas contain less than 1 mg As/kg dm [62 –64]. Some attention has been given lately to rice, because it is consumed often in large quantities and can contain up to 0.4 mg As/kg dm, in rare cases even 1 mg As/kg dm [65]. Inorganic arsenicals are often the major arsenic compounds in plant extracts [66, 67]. Some exceptions exist, where MA or DMA are dominating the arsenic speciation [68, 69]. There are only rare accounts on other arsenic species, for example TMAO or TETRA, in plants [70].
Urine is generally considered as the main excretion route of arsenic in mammals. The urine of unexposed humans usually contains around 5-10 µg As/L [71 –73]. The arsenic speciation of the urine of terrestrial mammals consists of 60-80 % DMA, 10-20 % MA (sometimes lower) and 10-20 % inorganic arsenic [74]. However, certain monkey species (marmoset, squirrel, tamarin), chimpanzees and guinea pigs are not able to metabolize arsenic, so that only inorganic arsenic is found in their urine [75, 76]. In contrast, it was recently discovered that urine of the giant panda contains almost only DMA, very little inorganic arsenic and hardly any MA, which marks this animal as a very efficient arsenic metabolizer [77]. There are also individual publications reporting the presence of trivalent methylated arsenicals [MA(III) and DMA(III)] in urine samples [78], but their accurate determination is very challenging because of their fast oxidation to their pentavalent equivalents [79, 80].
The liver is thought to be the main site of arsenic methylation in terrestrial mammals [81]. Transformation mechanisms have been proposed and investigated intensely. Already in 1945, Challenger published a possible arsenic pathway, consisting of alternating reduction and methylation steps, starting with arsenate and arsenite, going over trivalent and pentavalent MA and DMA and finally ending at TMAO and the volatile TMA (see Figure 5) [82].
14 Introduction
Figure 5. Classical Challenger pathway for arsenic biotransformation.
This classical Challenger pathway has been updated over the years, which lead for example to the pathway proposed by Hayakawa et al. in 2005 [83]. Here, arsenite is bound to glutathione and then consecutively methylated to yield MA(III) and DMA(III), which are quickly oxidized to MA and DMA. Other more or less modified versions of the classical Challenger pathway have been proposed [84]. There is general agreement that the arsenic methylation in terrestrial mammals is catalyzed by the arsenic (+3 oxidation state) methyltransferase, and that sulfur-containing chemicals like glutathione of S-adenosylmethionine are attached or bound to arsenic at some place during the transformation process [85 –87]. For a deeper insight into this topic, reading of the short, critical survey by Cullen is recommended [84].
In the marine environment, the dynamics of arsenic are completely different. For a start, seafood can contain up to about 100 mg As/kg dm, which is at least 100 times higher than the arsenic concentrations in most terrestrial living organisms [88]. The differences in the arsenic speciation patterns are even more striking: The main arsenic species in marine animals is arsenobetaine (AB), whereas algae predominantly contain arsenic in the form of dimethylated arsenosugars (see Table 3) [88 –91]. It has been proposed that algae take up inorganic arsenic from their environment and then transform it to arsenosugars. Further, these arsenosugars are metabolized to AB in marine animals or somewhere on the way from the algae to the animals. Several mechanisms have been suggested, but the actual biotransformation pathway(s) still remain undiscovered [92]. Arsenic compounds that are detected besides AB and arsenosugars can give important hints to the transformation mechanisms. Minor water soluble arsenic compounds that have been reported in marine samples are TMAO, AC, TETRA, DMAA, DMAP and trimethylated and also thiolated arsenosugars [91 –96]. Inorganic arsenic is usually very low in marine organisms, with a few exceptions like the edible seaweed Hijiki [97, 98]. Lipid-soluble arsenic species have been identified in marine samples, for example in fish oil [99, 100]. They have been studied intensively in the last years [51, 101, 102], but are still only known from the marine environment. Until now, the following arsenolipid classes have been detected: arsenic- containing hydrocarbons, fatty acids, long chain alcohols, phospholipids and phosphatidylcholines [50]. Additionally, phytyl 5-dimethylarsinoyl-2-O-methyl-ribofuranoside, which is an arsenolipid with a very unusual structure, was identified in an alga very recently [103].
Coming back to the formation of AB, it is often thought that the dimethylated arsenosugars of seaweed are either degraded to DMAE or converted to trimethylated arsenosugars, which are then degraded to AC. If DMAE is formed from the dimethylated sugars, it can either be oxidized to DMAA or
15 Introduction methylated to AC. Both compounds can be regarded as direct precursors of AB. It is also proposed that thio-analogues of the involved arsenic species could occur as intermediates (Figure 6) [94].
Figure 6. Proposed pathways for the formation of AB, adapted from [92, 94].
Experiments with seaweed showed that the originally present (dimethylated) arsenosugars are transformed into DMAE and further to DMA, MA and inorganic arsenic [104 –106]. Notably, neither AC nor AB was detected during these studies. However, it could be shown that AC is the major degradation product of trimethylated arsenosugars [107] and that AC is transformed to AB by bacteria and in mice [108, 109]. In the urine of sheep that were feeding on seaweed, containing high concentrations of arsenosugars, DMAA, DMAE and TETRA were detected in addition to the typical arsenic metabolites of terrestrial mammals (DMA as major compound, MA and inorganic arsenic) [110, 111]. It was also demonstrated that ingestion of arsenolipids from cod liver by humans leads to urinary excretion of thio-DMAP and thio-DMAB [112].
An alternative pathway was suggested by Edmonds, where arsenic is behaving similar to nitrogen during amino acid synthesis [113]. The core of the pathway is alkylation of DMA(III) by two-oxo acids,
16 Introduction leading to structures similar to methionine (Figure 7), where nitrogen is replaced by arsenic. For the formation pathway of AB, DMA(III) would have to react with glyoxylic acid to form DMAA (Figure 6).
More or less all of the hypothesized intermediate compounds have been found in the marine environment, but generally only at low concentrations [92, 95]. A different idea was seized by Maher et al., who point out the similarity between several marine arsenicals and organosulfur compounds that are occurring in the dimethylsulfoniopropionate and S-adenosylmethionine-methionine salvage metabolic pathways [95]. If such mechanisms exist for arsenic, a key intermediate compound would be arsenomethionine (an analogue of methionine where arsenic replaces the sulfur atom, Figure 7), which has not been detected so far. Of interest could be the detection of a compound called arsinothricin (Figure 7), which was produced from arsenite by the bacterium Burkholderia gladioli [114] and has a structure that could vaguely be correlated to arsenomethionine . Further, the detection of an organic polyarsenic compound, named arsenicin A (Figure 7), has to be mentioned [115]. Although its structure is fascinating, it was not detected in any other (marine) sample, which could simply be because its existence is often neglected.
Methionine Arsinothricin Arsenicin A Figure 7. Structures of methionine, arsinothricin and arsenicin A.
Several more water soluble arsenic species have been observed, albeit not in nature, but in a lab experiment by McSheehy et al [116] where inorganic arsenic was exposed to acetic acid and UV irradiation. The products included known arsenicals like AB, DMAA or AC, but also compounds that have not been reported anywhere else so far, including homologues of the before mentioned arsenicals with longer or shorter carbon-chains. The authors derived possible chemical structures of these arsenicals from the molecular mass and fragment masses obtained by electrospray ionization mass spectrometry [116].
Because of the structural similarity of AB to glycine betaine, which is a known osmolyte, it has been speculated and strongly indicated by experiments that AB can serve as an osmolyte as well [117]. Recent work by Hoffmann et al. has proven this very clearly for bacteria [109]. For a long time, it was thought that AB occurs exclusively in the marine environment. It took almost 20 years from the first discovery of AB in 1977 in lobster [89] until AB was also detected in terrestrial mushrooms in 1995 [38]. Further, TMAP, a homologue of AB, has been identified in marine organisms around two decades ago [118]. Since then, this species was occasionally found in other marine samples [119], but never in
17 Introduction the terrestrial environment. Its presence in extracts of hare muscle samples was suspected, but proof was lacking for a definite identification [120].
1.3.3 Arsenic species in macrofungi In the 1990s, quite a lot of work was dedicated to the arsenic speciation of mushrooms. This was probably triggered by the discovery that AB was the main arsenical in samples of the mushrooms Sarcodon imbricatus (scaly hedgehog, in German: Habichtspilz), Agaricus placomyces (nowadays known as Agaricus moelleri or inky mushroom) and Agaricus haemorrhoidarius (nowadays Agaricus silvaticus or Scaly Wood Mushroom, in German: Kleiner Wald-Champignon), see Figure 8 [38]. This was the first report ever of the occurrence of AB in a terrestrial organism. Until then, it was believed that AB exists exclusively in the marine biota.
A B
Figure 8. A: Sarcodon imbricatus, B: Agaricus silvaticus. A&B ©Jan Borovička .
Two years later, also for the first time in the terrestrial environment, reasonably high concentrations of AC and also traces of TETRA were detected in mushroom samples from old smelter sites [121]. Especially striking was the arsenic speciation of the well-known mushroom Amanita muscaria (fly agaric, in German: Fliegenpilz, Figure 9A), where AC accounted for more than 25 % of the total arsenic, making it the second-most abundant arsenical in these samples, after AB (60-70 %) [122]. Further, several unidentified arsenic species were found in methanol-water (9+1) extracts of A. muscaria with cation-exchange chromatography [122]. Another important step was the detection of an arsenosugar (the phosphate sugar) in a sample of Paxillus involutus (poison pax, in German: Kahler Krempling, Figure 9B) collected next to a gold mine [43].
Opposing the rich abundance of organoarsenicals in many mushrooms, there are also macrofungi that contain mainly (or sometimes even entirely) inorganic arsenic, for example Entoloma lividum (livid agaric, in German. Riesen-Rötling) [38], Boletus cavipes (in German: Hohlfuß-Röhrling) [123] or Lentinus edodes , better known as Shiitake [124]. Interestingly, lipid-soluble arsenicals have not been detected in mushrooms yet, which could be explained at least to some extent by their low lipid content [3].
18 Introduction
A B
Figure 9. A: Amanita muscaria, © Simone Braeuer. B: Paxillus involutus, ©Michaela & Gernot Friebes.
A review on arsenic species in various organisms was written by Dembitsky and Rezanka in 2003 [125]. It includes a large table that comprises the arsenic species in various mushrooms that were published at that time. In a broad study in 1997, the arsenic speciation of 38 fungal species was determined [44]. Most samples had AB as dominating arsenic compound, often accounting for 80 to 100 % of the sum of species. Inorganic arsenic and DMA often occurred at significant concentrations, occasionally even as main arsenical [inorganic arsenic: Amanita caesarea (Caesar’s mushroom, in German: Kaiserling) and Thelephora terrestris, DMA: some species of the genus Tricholoma (in German: Ritterlinge) and Volvariella volvacea (Straw mushroom, in German: Dunkelscheidiger Streifling)]. MA and TETRA were generally only detected at trace concentrations or even not at all. TMAO was not detected in any sample. The authors noted that the arsenic speciation is apparently not dependent on the lifestyle of the fungi, i.e. saprotrophic or mycorrhizal fungi. They speculated that AB is more abundant in higher evolved fungi and that more primitive fungal species therefore contain little to no AB, which is the case e.g. for mushrooms of the genus Tricholoma (for example T. sulphureum , the sulfur knight, in German: Gemeiner Schwefel-Ritterling, Figure 10A) [44].
In their publication on arsenic species in 73 samples of 46 different fungal species in 2014, Nearing et al. concluded that the rank in evolution is not the determining factor for the arsenic speciation in mushrooms [46]. They give as an example fungi of the Boletales order, which are highly evolved but contain in most cases DMA as main arsenical. Alternatively, the authors proposed that the arsenic speciation pattern is dictated by the fungal species and its morphology. They argue that for example gilled and puffball mushrooms (e.g. Lycoperdon perlatum or common puffball, in German: Flaschen- Stäubling, Figure 10B) contain predominantly AB, which could serve as an osmolyte, as already discussed earlier, and thus help keeping up the structure of the sporocarps. It was also observed that the substrate and the microbiota in the substrate seem to influence the arsenic speciation in sporocarps. Samples growing on wood did in general not contain significant concentrations of AB, whereas samples of the same species (e.g. Lycoperdon pyriforme) that were growing on soil contained the majority of the total arsenic as AB [46]. Overall, either AB, DMA or inorganic arsenic were the
19 Introduction dominating arsenic species in the 73 investigated samples. MA, TMAO, AC and TETRA were only detected occasionally and in most of these cases only as minor constituents of the mushroom’s arsenic speciation. Exceptions were for example Coprinus atramentarius (Inky cap, in German: Falten-Tintling) and Hebeloma velutipes (In German. Weißfleischiger Fälbling), where 40 % and 20 % of the extracted arsenic was TMAO, respectively. Another exception was a sample of Lactarius volemus (Weeping milk cap, in German: Brätling) which contained 22 % of the extracted arsenic as TETRA. Arsenosugars were only present in a few individual samples and at low concentrations [46].
A B
Figure 10. A: Tricholoma sulphureum, © Jan Borovička . B: Lycoperdon perlatum, © Michaela & Gernot Friebes.
Recently, the arsenic concentration and speciation of 19 mushroom samples obtained from local supermarkets in Yunnan province, China, were investigated [126]. Most samples contained less than 10 mg As/kg dm, with the exception of one sample of Lentinus edodes (42.3 ± 1.2 mg As/kg dm) and one sample of black boletus ( Boletus aereus , in German. Schwarzhütiger Steinpilz, 212 ± 5.7 mg As/kg dm) [126]. Inorganic arsenic turned out to be the main arsenical in many of the samples. AB and DMA were present in all of the samples, occasionally even as major arsenic compound. MA was only found in six of the 19 samples. Concerning the two mushrooms with the highest total arsenic concentrations, L. edodes primarily contained AB, accounting for over 90 % of the total arsenic, and over 90 % of the total arsenic in B. aereus was MA. Apparently, AC was not detected in any sample. The presence of other compounds like TMAO or TETRA was not addressed at all [126].
Concerning arsenic hyperaccumulators, Sarcosphaera coronaria contains more or less all arsenic as MA [38]. It is worth noting that there are only very few other accounts of MA in mushrooms at significant concentrations, which makes S. coronaria even more special. The second hyperaccumulating macrofungal species, Laccaria amethystina , contains between 70 and 97 % of the total arsenic as DMA [38, 40, 123], which was already discovered in 1991 [127]. It was noted that these high concentrations of the probably carcinogenic DMA can be a serious health risk for consumers of this “edible” mushroom [40]. The arsenic speciation of Lycoperdon perlatum , which is the third mushroom that is known to be able to contain more than 1000 mg As/kg dm, consists predominantly of AB, as already mentioned earlier [46].
20 Introduction
It is more or less accepted that the arsenic speciation depends on the fungal species. If two mushroom samples only belong to the same family or genus, this does not necessarily imply a similar arsenic speciation pattern. For example, the dominating arsenical in Laccaria amethystina is DMA, while Laccaria laccata (the deceiver, In German: Rötlicher Lacktrichterling) contains almost only inorganic arsenic [123].
To make things even more complicated, two samples of the same species can still contain different arsenicals. An interesting case is Sparassis crispa (cauliflower fungus, in German: Krause Glucke, Figure 11A), where one sample had AC as main and AB as second most abundant arsenic compound, whereas a second sample contained 45 % of the arsenic as AC and 45 % as an unknown arsenic species [44]. In a study on Imleria badia (bay bolete, in German: Maronenröhrling, Figure 11B), it was shown that the arsenic speciation in the fruit-bodies was heavily influenced by the substrate where the mushrooms where growing on. While samples from the vicinity of ironworks contained mainly inorganic arsenic, samples from close to a coal mine and sludge depositions contained at least 50 % of the arsenic as organic arsenic [47]. Unfortunately, the employed method (high performance liquid chromatography coupled to hydride generation atomic absorption spectrometry) did not allow the authors to specify this “organic arsenic” in any more detail.
A B
Figure 11. A: parassis crispa. B: Imleria badia, A&B: © Jan Borovička .
The detection of unidentified arsenic species in extracts of mushrooms has been reported several times [43, 122]. Recently, the arsenic speciation in the caterpillar fungus (yartsa gunbu, Ophiocordyceps sinensis , a parasitic fungus that grows out of its host insect larvae, Figure 12) was assessed in two publications [128, 129]. This mushroom is highly valued in traditional Chinese medicine and was found to contain up to 9 mg As/kg dm, which made consumers concerned about possible health risks [128]. While one article only found inorganic arsenic in extracts of around 10 %(v/v) nitric acid [129], the authors of the second article came to the conclusion that over 90 % of the total arsenic in these fungi were present as an unknown arsenical, eluting very early under anion-exchange conditions, close to arsenite [128]. During the work for the present doctoral thesis, an unknown compound with a similar retention behavior was observed as main arsenical in an extract of
21 Introduction
Tolypocladium ophioglossoides, which has also a parasitic lifestyle, like O. sinensis , but infects deer truffles instead of insects.
Figure 12. Ophiocordyceps sinensis, taken from [130].
Pure water or mixtures of water and methanol are usually well suited to extract most of the arsenic from mushrooms, but there are a few exceptions. One is the earth fan Thelephora terrestris , where pure water could only extract 8 % of the total arsenic, and methanol and water (9+1) only 2.5 % [44]. In another publication, extraction was carried out on T. terrestris by boiling the sample in water, which led to a much higher extraction efficiency and the detection of only inorganic arsenic in the extract [123].
Koch et al. simulated the bioaccessibility of arsenic in mushrooms from contaminated mine tailings, by extracting the samples with complex mixtures that should simulate the gastric and the intestinal environment [120]. A sample of Laccaria laccata was found to contain 46 mg As/kg dm, but only around 10 mg As/kg dm was extractable with this method. The main arsenic species were DMA and, surprisingly, TMAO. Samples of the genus Agaricus contained between 10 and 50 mg As/kg dm, and most of this was extractable, with AB as the main compound and in some cases also significant concentrations of inorganic arsenic [120].
Overall, it can be summarized that arsenic is easily extractable with aqueous solutions from most mushrooms. The majority of mushrooms that have been investigated so far contain AB, DMA or inorganic arsenic as main arsenical. Although very often one compound clearly dominates and accounts for up to 100 % of the total arsenic, some mushroom species also contain mixtures and have two or three main arsenic compounds. MA is only in very few cases the major arsenical of a mushroom and is otherwise only detected in traces or not at all. Beside these main and well-known arsenic species (inorganic arsenic, MA, DMA, AB), a variety of other arsenic species has been found in mushroom samples, although usually just at very low concentrations. These species are: TMAO, AC, TETRA and arsenosugars. It is not entirely understood what determines the arsenic speciation in mushrooms.
22 Introduction
Evidence exists that the fungal species is one important factor, together with the mushroom’s morphology and the substrate, but a clear proof is still needed.
1.3.4 Arsenic biotransformation in macrofungi? It is long known that microfungi have the ability to transform arsenic. Already in 1933, Challenger et al. discovered that the microfungus Scopulariopsis brevicaulis ( old name: Penicillium brevicaule ) processes inorganic arsenic into TMA [131]. From this observation, Challenger derived his well-known biotransformation pathway for inorganic arsenic [82], already discussed above. Besides S. brevicaulis , other microfungi have been found to transform arsenic as well [62].
In the case of macrofungi (mushrooms), it is an unsolved puzzle whether they are able to metabolize arsenic by themselves, or if they only take up different arsenic species from their environment. In the latter case, there are still a couple of possible different locations for the formation of arsenic, including the soil itself, plants and their interface with the fungi (the mycorrhizae) or microorganisms like bacteria or microfungi (see Figure 13).
Figure 13. Possible places of arsenic transformation.
It has been often demonstrated that microorganisms are able to degrade and also methylate arsenic species [125], including the formation of AB from precursors like DMAA [132]. On the other hand, if the arsenic biotransformation actually takes place in the macrofungi, the question remains if the entire fungal body possesses this ability or if only the mycelium fulfills this task and then transports the arsenic species to the fruit-body. Several studies have been conducted to solve this enigma. One of the
23 Introduction first in vitro experiments on arsenic in mushrooms was carried out in 1996 [133]. It was shown that fruit-bodies of the oyster mushroom Pleurotus sp . (in German: Seitlinge, Figure 14A) that were exposed to arsenate via the substrate contained almost exclusively inorganic arsenic (arsenate and arsenite), and only traces of MA. Further, mycelia of Agaricus placomyces (nowadays: A. moelleri , inky mushroom, in German: Perlhuhn-Champignon) were exposed to agar substrate with different arsenic species. Some of the provided MA was converted to DMA (16.8 % of sum of species), and AB and TETRA were taken up (but not converted) very efficiently by the mycelium, but no other conversions were observed [133]. This was surprising, because AB was found to be the main species in naturally grown fruit-bodies of this fungal species in an earlier study [38]. The authors noted that mycelia can reach a very old age in nature, which might influence the ability to transform arsenic species [133]. It has to be added that the experiment on Pleurotus sp. was carried out with quite high arsenic concentrations in the substrate (10 and 50 mg/kg dm), which is much higher than typical (bioavailable) arsenic concentrations in soil and might have led to a suppression of the arsenic transformation activities of the fungus. Similar effects were observed during in vitro experiments with Agaricus bisporus (the famous white button mushroom, In German: Champignon, Figure 14B), where treatment with high concentrations of arsenate, namely 100 mg As/kg solutions, seemed to inhibit the transformation of arsenic [134]. Most of the arsenic in the fruit-bodies was present as inorganic arsenic, while fruit- bodies that were exposed to lower doses of arsenate (10 mg As/kg) contained also significant concentrations of DMA and AB.
A B
Figure 14. A: Pleurotus ostreatus. B: Agaricus bisporus. A&B: ©Jan Borovička .
Continuing with the investigation of the source of arsenic speciation in mushrooms, Soeroes et al. cultivated A. bisporus on wheat straw mixed with horse dung [135]. AB was the main compound in the fruit-bodies, but was not detected in the substrate. MA was also only found in the fruit-bodies and not in the substrate. In contrast to this, inorganic arsenic and DMA were both found in fruit-bodies as well as in the substrate. TMAO was only present in the substrate. When the substrate was treated with 1000 mg As/kg of arsenate, the total arsenic concentration of the mushrooms was much higher than in the control (22.8 instead of 0.5 mg As/kg dm). Although DMA and MA concentrations were higher
24 Introduction in the treated samples than in the untreated ones, their relative abundance was much lower, and 98 % of the extractable arsenic was inorganic arsenic [135].
In another study with A. bisporus, the mushrooms were grown on “mine waste material” and in parallel on arsenate [136]. The total arsenic concentration in the substrates was between 180 and 360 mg As/kg dm. When no fungal mycelium was added, the substrates only contained TMAO and inorganic arsenic. In mushrooms, large quantities of AB were found, besides DMA, inorganic arsenic and an unknown compound. It was noted that also the mycelium contained AB. This indicates that the microbial communities in the substrates are not solely responsible for the formation of AB [136].
More recently, the fate of arsenic during the vegetative (i.e. mycelium only) and the reproductive life stage (i.e. mycelium with fruit-body) of fungi was investigated [134, 137]. First, samples of three different fungi [ A. bisporus (Figure 14B), Suillus luteus (Slippery jack, in German: Butterpilz, Figure 15) , and Sparassis crispa (Figure 12)] were exposed to different arsenic compounds, and then the mycelium was investigated for its arsenic speciation [137]. It turned out that in all but one sample, only the one arsenic species that was added to the substrate was present in significant concentrations in the mycelium. In one sample of S. crispa , however, arsenate was partly converted to TMAO (around 10 % of the total arsenic). From these results, it was speculated that not the fungi, but rather microorganisms or a selective uptake of the arsenic species from soil are responsible for the arsenic speciation in mushrooms [137].
Figure 15. Suillus luteus © Michaela & Gernot Friebes.
The same authors also studied the arsenic speciation in Agaricus spp. during fruit-body production (reproductive life stage) [134]. It could be shown that AB was neither detectable in the mycelium nor the substrate before the development of fruit-bodies. However, from the moment of first appearance of young fruit-bodies, the so-called primordia, and onwards, fruit-bodies and even the underlying mycelium/substrate mixture contained significant concentrations of AB, besides inorganic arsenic and DMA. In the same study, arsenic species of small portions of the interface area between fruit-body and substrate were mapped with µXANES [134]. This experimental setup revealed that the substrate contained only inorganic arsenic, whereas AB and also DMA or TMAO (not distinguishable with the 25 Introduction applied method) were only detectable in the fungal tissues. The authors concluded that it is still not entirely resolved whether the arsenic species are formed by the fungus itself or by microorganisms in the substrate, from where it is then quickly transported to the fungal tissues [134]. Still, the results indicate that the transformation of arsenic is linked to the reproductive life stage of the fungi.
As can be seen, research on the arsenic biotransformation in mushrooms has dealt more or less only with the question of the active metabolizers, but not with the transformation mechanisms themselves, as is the case for the marine environment. It is often assumed that the formation of AB that is present in mushrooms is analogous to the marine biotransformation pathway of AB. However, the low abundance of arsenosugars in mushrooms makes this unlikely, because they would be needed as initial compounds [46].
1.4 Toxicity and regulations on arsenic Arsenic is known for its acute and chronic toxicity [138]. The acute lethal dose of inorganic arsenic is as low as 100 - 200 mg [139]. Chronic exposure to inorganic arsenic can lead to skin lesions, diabetes and several types of cancer [138]. Organic arsenicals are in general much less toxic than inorganic arsenic, with a few exceptions. For example, MA and DMA are classified as group 2B carcinogens (possibly carcinogenic) [140]. Further, trivalent arsenic species like MA(III) and DMA(III) are considered much more toxic than their pentavalent equivalents. MA(III) has shown even higher toxicity than arsenite [141, 142]. On the other hand, AB is nontoxic at all and is excreted unchanged by mammals [143]. Also the compounds AC and TMAO did not display any significant toxicity [91, 144, 145]. However, some toxic activity was reported for TETRA [146]. In the case of arsenosugars, no direct toxicity was observed, but their biotransformation in humans can result in toxic products [147]. Finally, during the last years, it has been shown that lipid soluble arsenicals, such as arsenic containing hydrocarbons, are probably also highly toxic [54, 148].
To minimize health risks from the ingestion of arsenic, official regulations have been established for maximum concentrations of total arsenic or inorganic arsenic in food and drinks [149]. Already in 1993, the World Health Organization has suggested 10 µg/L as a maximum limit for arsenic in drinking water [55]. This has been implemented by several (inter)national agencies like the European Union [150] or the U.S. Environmental Protection Agency (US EPA) [151]. In 2015, the European Commission set maximum levels for inorganic arsenic in rice and rice products, ranging from 0.1 mg As/kg in infant food to 0.3 mg As/kg in rice waffles etc. [152]. At the moment, the only country with an official limit for arsenic in fungi is China (0.5 mg/kg) [153]. The European Commission has only set limits for lead and cadmium in fungi (0.3 mg Pb/kg and 0.2 mg Cd/kg for the most commonly grown mushrooms and 1 mg Cd/kg for other mushrooms) [154].
26 Introduction
There was a provisional tolerable weekly intake maximum of 15 µg As/kg body mass (bm) established by the Joint FAO/WHO Expert Committee on Food Additives [155], but this has been withdrawn in 2011 [156]. Further, so-called minimal risk levels (MRLs) have been set up by the Agency for Toxic Substances and Disease Registry for different arsenic species [157]. The MRL is defined as “an estimate of daily human exposure to a substance that is likely to be without an appreciable risk of adverse effects over a specified duration of exposure”, but does not account for carcinogenic effects [140, 157]. In the case of inorganic arsenic, the MRL for acute duration (up to two weeks) is 5 µg/kg bm/day and the MRL for chronic duration (more than one year) is 0.3 µg/kg bm/day. The MRL (chronic duration) for MA is 10 µg/kg bm/day and for DMA it is 20 µg/kg bm/day [140, 157].
Finally, the health risk can be estimated with the help of the benchmark dose lower limit (BMDL) and the margin of exposure (MoE). For example, the US EPA has derived from animal studies a BDML 10 (meaning a 10 % risk increase) of 430 µg/kg bm/day for DMA [158]. This BMDL has to be divided by the daily exposure to give the MoE. For a BMDL 10, an MoE of at least 10 000 is considered safe [159].
1.5 Techniques for arsenic speciation analysis in environmental samples Several techniques can be employed for the determination of total arsenic concentrations in environmental samples. The most commonly used ones are inductively coupled plasma mass spectrometry (ICPMS), inductively coupled plasma optical emission spectroscopy (ICPOES), atomic absorption spectroscopy [AAS, with or without graphite furnace (GF) or hydride generation (HG], atomic fluorescence spectroscopy (AFS) and neutron activation analysis (NAA) [13]. The methods are well established and routinely used around the world.
The determination of arsenic species is not as common as total element analysis and can be prone to errors and misinterpretations if not thoroughly studied and understood. Applied techniques include: separation via liquid chromatography and then detection via ICPMS or molecular mass spectrometry [most often with electrospray ionization (ES-MS) and less commonly atmospherical pressure chemical ionization (APCI)], capillary electrophoresis (CE) coupled to ICPMS or ES-MS, X-ray absorption spectroscopy techniques such as X-ray absorption near edge structure (XANES) or extended X-ray absorption fine structure (EXAFS), and to a certain degree HG-AAS, HG-ICPMS or gas chromatography (GC) coupled to ICPMS. Of course, all of the mentioned techniques have advantages but also drawbacks.
Going back in time, the first work on arsenic speciation analysis was reported by Braman and Foreback in 1973 [160]. They took advantage of the facts that different arsenicals can be reduced to the corresponding arsines at different pH values (arsenite: pH 4-9; arsenate, MA and DMA: pH 1-2), and that different arsines have different boiling points (arsine: -55°C, methylarsine: 2°C, dimethylarsine: 36°C; TMA: 52°C) [80]. The experiment involved HG (reduction to arsines) and then cold (or: cryogenic) 27 Introduction trapping of the arsenicals before analysis with a “heat scanning monochr omator-photometric readout system ” [160].
Nowadays, HG-AAS (with or without cryotrapping) is occasionally used for arsenic speciation analysis [161, 162]. However, it can only be used for molecules that can be reduced to a volatile compound and is thus not suited for arsenic species like AB or AC. Still, it profits from a very good sensitivity because of the almost matrix-free measurement. The short analysis times make it a good option for labile arsenic species.
In 1999, three different detectors (ICPMS, AAS and GF-AAS), combined with anion-exchange liquid chromatography, were compared for arsenic speciation analysis in mushroom extracts [123]. ICPMS had an approximately 1000 fold better limit of quantification (0.1 ng when the injection volume was 100 µL, thus 1 µg/L) than flame AAS and GF-AAS. This means that the two AAS methods can only be used for the quantification of high concentrated arsenic species. Further, it is not possible to produce a continuous signal with GF-AAS, and the time-resolution was around 70 seconds per data point. In order to be still able to distinguish between the different arsenicals, the LC separation method was adapted and one chromatogram took 50 instead of 6 minutes [123].
AAS and also AFS can be improved by introducing a decomposition step to arsenate and then reduction to arsine after separation by HPLC. With this setup, HG-AFS can detect arsenic at sub µg/L concentrations and almost rivals the detection limits of ICPMS and is also not limited to hydride- forming arsenicals anymore [80, 163]. The low purchase and operating costs in comparison to ICPMS make HG-AFS an attractive alternative to ICPMS [80].
Another technique that is limited to volatile compounds is GC-ICPMS. A common workaround is the derivatization to volatile species, but this can lead to difficulties in identifying the structures of the original compounds. Still, the “dry” plasma conditions (no liquid mobile phase) can lead to improved sensitives, and less plasma energy is needed, because there is no need for drying and vaporizing [164]. Hyphenating GC and ICPMS can prove to be tricky, because special transfer lines between the outlet of the GC and the torch of the ICPMS are necessary to ensure a constant temperature [165]. In general, GC is rarely used for the separation of arsenic species [80].
Although some publications on CE separation of arsenicals exist, poor limits of detection and difficult applicability to real samples limit its uses [80]. The coupling to ICPMS is tricky, because buffer concentrations and the ionic strength should be kept very low to avoid crystallization on the nebulizer of the ICPMS. Additionally, designing a good interface is difficult because the electrical contact has to be maintained all the time to ensure a stable electrical current for the separation, and also the flow rates of CE and nebulizer have to be adjusted (from a few nL/min to some µL/min) [165]. Both
28 Introduction requirements are usually fulfilled with the introduction of a sheath liquid [166]. Clear advantages of CE-ICPMS are its high resolution and that positive, negative and neutral species can be separated in one single run [167]. The low injection volumes can be regarded both as advantage, because only very little sample is needed, and also as drawback, because it leads to high limits of detection (typically some µg/L) [166 –168]. Gel electrophoresis (1D or 2D) has been used in combination with laser ablation - ICPMS, for example for the investigation of complex protein mixtures. This suffers from the often low purity of biochemical reagents concerning the elements of interest, which leads to an increased background [165].
All of the techniques discussed so far are designed for the analysis of liquid samples. For solid samples, this means that an extraction step is required prior to analysis. However, the selection of the extracting solution can influence the results significantly, for example by a selective extraction of only a part of the arsenic speciation (e.g. water-soluble vs. lipid-soluble arsenicals), or by reactions of the extractant with the arsenic compounds, which would change the species completely. A huge problem is the stability of trivalent and sulfur containing arsenic species, which are oxidized quickly in solution and could be misidentified as their pentavalent and oxygenated equivalents.
These problems can be avoided by using solid-phase analysis techniques like X-ray absorption spectroscopy, for example XANES or EXAFS. These techniques can deliver valuable information about neighbor atoms of a target element [80]. Even when synchrotron-based radiation is employed to gain sensitivity, the limits of detection are still very high, namely in the range of 1-10 mg/kg [167]. Also, only compounds that account for at least 5 % of the total arsenic can be identified, and standards have to be available for identification [169]. Additionally, two or more arsenic species where arsenic has the same neighboring atoms cannot be distinguished, which is the case for example for AB, AC and TETRA, which makes the gain of information through these techniques very limited [80]. X-ray absorption spectroscopy is sometimes used as a complementary method to HPLC-ICPMS. It can also be applied for imaging the arsenic species distribution in solid samples, where a spatial resolution of a few µm can be achieved with µ-XANES [134, 170].
Still, the most popular technique for arsenic speciation analysis is HPLC-ICPMS, very often with ion- exchange methods [80]. It benefits from its excellent detection limits (as low as 10 ng As/L, depending on the injection volume), a high dynamic range and the compound independent response [167, 171]. This allows the quantification of arsenic species even if their structure is unknown. Another bonus is the easy connection of HPLC and ICPMS, as no special interface like in CE or GC is necessary [170]. Attention has to be given to the so-called carbon enhancement effect, which leads to an increase of the arsenic signal when higher amounts of carbon (e.g. from an organic mobile phase or extracting solution) are present in the plasma [172, 173]. If ignored, this effect can lead to serious
29 Introduction misinterpretations concerning the concentrations of individual arsenicals. However, this effect can also be used beneficially to increase the arsenic sensitivity and thus lower the limit of detection [163]. Arsenic is naturally occurring as a monoisotopic element with an atomic mass of 75. Its analysis with ICPMS suffers from the polyatomic interference ArCl +, but this can be counteracted by introducing a collision gas like helium into the collision/reaction cell before the quadrupole mass filter to remove the interference. Alternatively, a reactive gas like oxygen or methyl fluoride can be added that reacts with arsenic so that it can be detected at a mass shift, away from the interference. This is efficiently achieved with triple quadrupole instruments [174, 175]. Another possibility to deal with interferences is the use of high resolution ICPMS, where arsenic and its interferences can be distinguished from each other by their exact masses [170].
The downside of HPLC-ICPMS is that not much can be said about the structure of such unknown compounds. Although the behavior at different chromatographic conditions can give clues on its chemical properties (e.g. polarity, charge at different pH levels, size), the final identification of a new species has to be fulfilled with another technique. In addition, the use of organic mobile phases, which is generally required for reversed phase chromatography, necessitates certain adaptions to the ICPMS and a very careful handling [176]. The introduction of high amounts of organic solvents to the ICPMS can cause instabilities and even extinction of the plasma [164]. Measures to keep up a stable plasma include for example the reduction of the flow rates from the HPLC, cooling of the spray chamber below 0°C or installation of a desolvation unit [165]. To avoid carbon deposition on the interface, oxygen has to be added to the gas flow [164]. Even if all these actions can be tedious work, it facilitates the combination with ES-MS immensely.
Molecular mass spectrometry, most commonly ES-MS and less frequently APCI-MS, with previous separation by HPLC is quite complementary to ICPMS. It can deliver the molecular mass of an unknown compound, and data on fragmentation allows insight into the chemical structure of an analyte and can even lead to the identification of new compounds [177 –179]. High resolution MS or MS/MS instruments can be very valuable for the identification of unknown compounds [169, 170]. ES-MS is well suited for the analysis of highly polar or ionic analytes and macromolecules with multiple charges, such as proteins [166]. Drawbacks of ES-MS are that the technique suffers from matrix effects, such as ionization suppression of the analyte, and that quantification can be challenging [80, 167]. Even more problematic are the compound dependent response, due to different ionization efficiencies, and the limits of detection, which are highly depending on the analyte. While the permanent cations AC and TETRA can be detected at concentrations as low as 1.5 µg As/L, the limits of detection are much higher for rather anionic compounds like arsenate or MA (around 200 µg As/L) [178]. Furthermore, limits of detection can be significantly influenced by the matrix, which can turn out to be a considerable problem for the investigation of environmental samples without previous cleanup steps. Here, MS/MS 30 Introduction experiments can be a helpful tool [180]. ES-MS is not element specific and thus will show not only arsenic containing compounds, but also everything else in the sample. This is less problematic when pure compounds are investigated, but poses a major difficulty for natural samples. Even if mushrooms with record arsenic concentrations are used (roughly in the order of 1000 mg/kg dm), this accounts only for around 0.1 % of the dried sample and can easily get lost beneath major constituents of the mushroom like carbohydrates, proteins or lipids.
This difficulty is illustrated effectively by an example from this thesis: In Figure 16, chromatograms of a standard containing 10 µg As/L of AB, TMAO, AC and TETRA, run under the same chromatographic conditions, detected with ICPMS and and ES-MS as are shown (injection volume: 10 µL). It can be seen that these cationic arsenicals are detected on ES-MS with a good sensitivity in this pure standard solution.
Figure 16 B: Cation-exchange chromatograms of a standard with 10 µg As/L of AB, TMAO, AC and TETRA with ICPMS (7700, m/z 75 in helium collision gas mode) and ES-MS [SIM mode, m/z 179 (AB +), 137 (TMAO+H +), 165 (AC +) and 135 (TETRA +)] as detectors. Note that ES-MS signal is on the secondary y-axis and starts at 4000, but range is the same for both axes (0-20000 and 4000-24000). Retention time offsets are due to different HPLC instruments and different delay volumes between column and detector; e.g. because a UV detector was included in front of ES-MS.
However, when looking at a mushroom extract ( Ramaria cf. fagetorum) in Figure 17, things get more complicated. In the selected ion monitoring (SIM) mode [m/z: 179 (AB +), 137 (TMAO+H +), 165 (AC +) and 135 (TETRA +)], the arsenic species that were detected with ICPMS are still visible, but other peaks are also present, very likely due to compounds that also have one of the selected molecular masses, but are not containing arsenic. In the case of unknown arsenicals, it gets even worse, because their molecular masses are not known and ES-MS has to be operated in scan mode, which is shown in Figure 17B. Without any reliable reference from ICPMS, detection of arsenic containing compounds is more or less impossible.
31 Introduction
This demonstrates effectively the differences between ICPMS and ES-MS. The two techniques can complement each other in a very convenient way for the investigation of unknown arsenic species. They hav e even been called the “dream team in the life sciences” [181]. ICPMS is essential to focus the selection of ions, since it only shows arsenic-containing compounds, and allows the quantification of unknown arsenicals. On the other hand, ES-MS can deliver information on the molecular mass and on the chemical structure of unknown compounds, and it can be useful to detect co-eluting compounds.
Figure 17. Cation-exchange chromatograms of a Ramaria cf. fagetorum extract. A: ICPMS (7700, m/z 75 in helium collision gas mode) and ES-MS in SIM mode [m/z 179 (AB +), 137 (TMAO+H +), 165 (AC +) and 135 (TETRA +)]. B: ES-MS in scan mode (m/z 40-400), note that the scaling y-axis of B is 10 times larger than of A.
Traditionally, unknown arsenicals are first detected with HPLC-ICPMS, then fractions that contain these unknown compounds are collected and often also concentrated, and finally these fractions are subjected to ES-MS [170]. However, both techniques can also be used simultaneously: After separation by HPLC, the effluent is split and one part is introduced to the ICPMS, while the other part is guided to the ES-MS [182, 183]. The advantages of this setup are that it is less time-consuming and that the species are detected by ICPMS and ES-MS at the same time, which is especially interesting for labile
32 Introduction compounds [170]. However, a very sophisticated instrumental setup is needed. It has also to be kept in mind that it requires a mobile phase that is compatible with both detection systems [163, 167]. While ES-MS needs volatile mobile phases with a low surface tension and not easily ionized buffers, ICPMS has problems with too high amounts of organic mobile phases and prefers solvents with a low molecular weight [169, 184].
33 Overview of the publications
2 Overview of the publications
For this thesis, three papers on arsenic in mushrooms were published:
Publication 1: Braeuer S, Goessler W, Kameník J, Konvalinková T, Žigová A, Borovička J (2018) Arsenic hyperaccumulation and speciation in the edible ink stain bolete ( Cyanoboletus pulverulentus ). Food Chem. 242:225 –231 (see Figure 18)
Figure 18 . Cyanoboletus pulverulentus. ©Jan Borovička .
Publication 2: Braeuer S, Borovička J, Goessler W (2018) A unique arsenic speciation profile in Elaphomyces spp. ("deer truffles") - trimethylarsine oxide and methylarsonous acid as significant arsenic compounds. Anal. Bioanal. Chem. 410(9):2283 –2290 (see Figure 19)
Figure 19 . Elahomyces asperulus. ©Jan Borovička .
Publication 3: Braeuer S, Borovička J, Glasnov T, Guedes de la Cruz, G, Jensen KB, Goessler W (2018) Homoarsenocholine - a novel arsenic compound detected for the first time in nature. Talanta (188):107 –110 (see Figure 20)
Figure 20 . Ramaria subbotrytis. ©Jan Borovička .
34 Overview of the publications
In publication 1, the edible Cyanoboletus pulverulentus (ink stain bolete, in German: Schwarzblauender Röhrling) was identified as arsenic hyperaccumulating species. 39 samples were collected and investigated, along with soil samples from the collection sites. It turned out that the arsenic concentration in the fruit-bodies is not related at all to the arsenic concentration in the underlying soil. Total arsenic in C. pulverulentus occurred in a very broad range, from 2.4 to 1300 mg As/kg dm, with a median of 160 mg As/kg dm. Since this mushroom is collected and eaten by connoisseurs, a possible health risk for consumers was evaluated. In order to do this, the arsenic speciation of C. pulverulentus was determined with HPLC-ICPMS. Extraction was carried out with water. With this method, around 80 % of the total arsenic was extractable. Column recoveries were more or less quantitative (78 – 110 %). The only arsenic species that was found in the aqueous extracts was DMA, besides some occasional traces of MA. DMA is considered a possible human carcinogen. Because of this, the benchmark dose lower limit (BMDL10) and the margin of exposure (MoE) were calculated. Based on the results, we do not recommend the consumption of C. pulverulentus on a regular basis, because already the ingestion of as little as 90 g per year can increase health risk significantly. It has to be pointed out that these considerations assume a chronic exposure, meaning a continuous intake of this mushroom over many years, which is probably not very likely for the general public.
Publication 2 addresses arsenic in three species of the genus Elapohmyces (deer truffles, in German: Hirschtrüffel), which are hypogeous macrofungi. They can accumulate remarkable arsenic concentrations up to 660 mg As/kg dm. By definition, this does not make them hyperaccumulators, but they are still amongst the top arsenic accumulators amongst macrofungi. Within the Elaphomyces samples, the arsenic concentration depended on the fungal species, with 120 – 600 mg As/kg dm in E. granulatus and E. muricatus , but only 12 – 42 mg As/kg dm in E. asperulus . Surprisingly, the extraction efficiency was much better for E. granualtus and E. muricatus (around 80 %) than for E. asperulus (3 – 14 %). These differences are demonstrated clearly in Figure 21. It is worth mentioning that one sample was first identified as E. asperulus . Since the total arsenic concentrations and the extraction efficiency were much higher than in the other samples of this species, the fungal species was determined again by our colleagues, and it turned out that it was indeed E. granulatus . Unfortunately, this confirmation was only possible after submission of the manuscript for publication, so that this ambiguous sample was not included in the paper. Another interesting result on the side was that the concentrations of the alkali elements sodium, potassium and rubidium (and to a small extent caesium) were also significantly lower in E. asperulus than in the two other species. The cases of arsenic concentration, extraction efficiency and concentrations of alkali elements demonstrate nicely how element concentrations could be a valuable additional tool for fungal taxonomic characterizations.
35 Overview of the publications
800 100
80 600
60
400
40 Extracted As [%] Total As [mg/kg As Total dm] [mg/kg 200 20
0 0 ASP-44 ASP-55 ASP-57 ASP-84 ASP-56 ASP-58 ASP-59 ASP-85a ASP-85b E. granulatus E. muricatus E. asperulus
Figure 21. Arsenic concentrations and extraction efficiencies (secondary y-axis) of Elaphomyces spp.
While the results of arsenic speciation analysis were extremely simple in publication 1 (just one arsenic species) and served primarily for the estimation of a possible health risk, arsenic speciation analysis turned out to be more complex in the samples of publication 2. To begin with, the main arsenical in the deer truffles was MA. This compound is usually only present at trace concentrations in mushrooms, or often cannot be detected at all. There are only very few fungal species where MA has been reported as main arsenical. Notably, one of them was Sarcosphaera coronaria . This fungus belongs to the division of ascomycota, which is also the case for the genus Elaphomyces . In contrast, most of the other investigated mushrooms so far belong to the division of Basidiomycota. For example, in the large study by Nearing et al., there was only one ascomycete included. It was a sample of Morchella esculenta , where 100 % of the arsenic was present as inorganic arsenic [46].
Coming back to Elaphomyces , another interesting discovery was the high concentrations of TMAO (up to 37 %), which is also only rarely found in other macrofungi. Finally, we were also able to detect significant concentrations of MA(III). This is the first report on the presence of this arsenical in mushrooms. It is highly toxic, but deer truffles are generally not consumed by humans. Wild boars, however, are actively searching for these macrofungi. One idea that was raised within this publication is that Elaphomyces fungi use arsenic to attract animals. Since these fungi are growing underground, they rely on animals to spread their spores (e.g. via the feces). TMAO can easily be reduced to the volatile TMA, which is supposed to have a quite distinctive smell and could be the attractant for the boars. This would be an explanation for the high concentrations of TMAO in deer truffles, but thorough studies will have to be conducted to proof this idea.
Publication 3 is purely focusing on the arsenic speciation, and the total arsenic concentration is only a minor point. For this paper, so-called coral mushrooms of the genus Ramaria were investigated. They 36 Overview of the publications are very unusual terrestrial organisms, resembling marine corals. The main arsenic species in all samples was AB, accounting for around 84 ± 9 % of the extracted arsenic. This is also the case for many other mushrooms and therefore not very remarkable. Much more extraordinary are the other arsenic species that are occurring in all investigated Ramaria samples at not very high but still easily measurable concentrations. Many of these compounds have not been identified yet. Some of these unknown compounds are well retained under anion-exchange (Figure 22) and others under cation- exchange conditions (Figure 23). Luckily, enough mushroom material was available, and so the identification of one of the unknown arsenic species was tackled. The unknown compound that eluted from the cation-exchange column right after TETRA was selected for this task (UNK A). The highest concentration of it in the samples was 300 µg As/kg dm. Due to the dilution through extraction, the concentration in the solution was only around 15 µg As/L. Since this is very low for ES-MS analysis, the extract was injected multiple times onto the cation-exchange column, and the fraction containing the UNK A was collected (see experimental section). The fraction was concentrated and then subjected to ES-MS analysis.
25 unretained As DMA 20
15
10 DMAA
Intensity [kCPS] 5 ? MA ? ? As (V) ? 0 0 2 4 6 8 10 12 Time [min]
Figure 22. Anion-exchange chromatograms of a Ramaria extract.
250 unretained As AB
200
150 AC 100
Intensity [kCPS] TMAP 50 TMAO ? TETRA UNK A ? ? 0 0 2 4 6 8 10 Time [min]
Figure 23. Cation-exchange chromatograms of a Ramaria extract.
37 Overview of the publications
With ES-MS, it was possible to detect a compound with m/z 179 at the retention time of UNK A. Because of its retention behavior, it was assumed that this compound must be very cationic, probably a permanent cation like AC or TETRA. In addition, it was well extracted with pure water, which means that it has to be a water-soluble species and no lipid. The molecular masses of several possible candidates were calculated, and a homologue of AC, with one more carbon in the chain, was a perfect match (Figure 24).
Figure 24. Homoarsenocholine (AC2).
The exact mass was calculated (179.0411), and the collected fraction was investigated with high resolution MS (“Orbitrap”). The exact mass was confirmed, and characteristic fragments that perfectly suited the proposed structure were detected through fragmentation experiments. The arsenic species (3-hydroxypropyl) trimethylarsonium, which we named homoarsenocholine (AC2), was synthesized by colleagues as described in the supplementary material of the publication. Its identity was confirmed through NMR. The pure compound was dissolved in water and then investigated with HPLC-ICPMS and HPLC-high resolution MS, similar to the fraction of the mushroom extract. Finally, the mushroom extract was co-chromatographed with the pure compound. Thus, the presence of AC2 in Ramaria mushrooms was confirmed.
Curiously, AC2 had already been mentioned in a publication before. In 2005, McScheehy et al. conducted experiments where they mixed inorganic arsenic with acetic acid and let them react under UV irradiation [116]. One of the detected products was AC2. The authors reported the same characteristic fragments that we found as well. Apart from this publication on an in vitro experiment, this is the first discovery of AC2 in a natural sample. As a bonus, we also detected DMAA and TMAP in all of the Ramaria samples. To the best of our knowledge, they have never been found in any terrestrial natural sample before.
The presence of AC2, DMAA and TMAP in mushrooms raises the huge question about the biotransformation mechanism(s) of arsenic. DMAA is an intermediate in some of the proposed pathways on the formation of AB, but TMAP and AC2 are never mentioned. Possible next candidates for identifying unknown arsenicals are aldehydes, which are typical intermediates between alcohols and acids. There is only one old publication that mentions the detection of AB aldehyde [185], but from then on, such compounds were not investigated or reported any more.
38 Overview of the publications
Apart from aldehydes, it would also be fascinating to find trimethylated arsenosugars in mushrooms, because this would deliver evidence of one of the hypothesized transformation pathways (Figure 6). These compounds have been found in marine samples, but only at low concentrations.
It will require much more work and the identification of other still unknown arsenic species to elucidate this mystery.
39 Experimental
3 Experimental
All mushrooms were thoroughly cleaned with water and then freeze-dried. They were then homogenized with a rotary mill, equipped with a 1 mm sieve and rotor made from titanium.
The concentrations of 35 elements were determined in all samples. For this, aliquots of the samples (usually around 100 mg, weighed to 0.1 mg) were digested with 5 mL nitric acid in a microwave-heated autoclave (Ultraclave III or IV, MLS GmbH, Leutkirch, Germany). The loading pressure was 40 bar (Argon 5.0, Messer). It was heated up to 250°C and then held there for 30 minutes. After cooling down, the samples were filled into 50 mL polypropylene tubes (Greiner bio-one, Kremsmünster, Austria) and diluted with ultrapure water (18.2 MΩ*cm, Millipore, Bedford, USA) to a final acidity of 10 % v/v. Each sample was prepared in triplicates. For quality control, aliquots of the standard reference material (SRM®) 1573a (Tomato leaves, NIST, Gaithersburg, USA) were digested together with the samples (usually three replicates in one digestion with 40 vessels). Digestion blanks were prepared as well.
The calibration standards were prepared from single element standards. The calibration levels were chosen to fit best the expected concentrations in the digests. It was decided to split the elements into two calibration groups because of the large concentration differences between the highest and the lowest concentrated elements, in order to avoid contamination by trace impurities of the higher concentrated standards (Table 4 and Table 5).
Table 4. Calibration A. Prepared in 10 % v/v nitric acid ((≥ 65%m/m p.a., Carl Roth GmbH + Co.KG, Karlsruhe, Germany, further purified via sub-boiling). All concentrations in [µg/L].
Elements 1 2 3 4 5 6 7 Bi, Gd, Mo, Sb, Te, Tl, U 0.001 0.005 0.01 0.05 0.1 0.5 1 Ag, B, Ba, Cd, Ce, Co, Li, Ni, Pb, Se, Sn, Sr, V 0.01 0.05 0.1 0.5 1 5 10 As, Cr, Cs, Mn 0.1 0.5 1 5 10 50 100 Al, Cu, Fe, Rb, Zn 1 5 10 50 100 500 1000
Table 5. Calibration B. Prepared in 8 % v/v nitric acid and 2 % v/v hydrochloric acid (sub-boiled). All concentrations in [mg/L].
Elements 1 2 3 4 5 6 7 8 Hg (*10 -3 ) 0.01 0.05 0.1 0.5 1 5 10 - Ca, Mg, Na 0.01 0.05 0.1 0.5 1 5 10 - P, S 0.01 0.05 0.1 0.5 1 5 10 50 K 0.1 0.5 1 5 10 50 100 200
40 Experimental
It has to be noted that this thesis is part of a larger project. Within this project, many different mushrooms were analyzed, and most of them have never been investigated for their element concentrations before. Since some mushrooms can accumulate various elements, and others not, a very wide calibration range was chosen. After the measurement, the results were carefully examined, and unnecessary high calibration standards were excluded from the calibration.
For each measurement, the SRM(R) 1640a (Trace elements in water, NIST) was prepared one time by diluting it 1+9 (10 % v/v nitric acid). An internal standard solution, consisting of approximately 200 µg/L Be, Ge, In and Lu, was added online to the samples via a T-piece before the nebulizer (Tubing material: Tygon®; inner diameter of sample tubing: 1.02 mm; inner diameter of internal standard tubing: 0.19 mm).
For the measurements, either a triple quadrupole ICPMS (ICPQQQMS 8800) or a ICPMS 7700 (both from Agilent Technologies, Waldbronn, Germany) instrument was used. The selected m/z values and the octopole gas modes are listed in Table 6.
41 Experimental
Table 6. Selected octopole gas modes and m/z ratios for each element that was determined. O2-modes were only with ICPQQQMS. *preferred tune mode with ICPMS 7700. **preferred tune mode with ICPQQQMS 8800.
Element Octopole gas mode m/z
Li nogas 7 B nogas 11 Na He 23 Mg He 24 Al nogas 27 P Nogas, He*, O2** 31, 31, 31 -> 47 S Nogas, He*, O2** 34, 34, 32 -> 48 K He 39 Ca He 43 V He 51 Cr He 52 Mn He 55 Fe He 56 Co He 59 Ni He 60 Cu He 65 Zn He 66 As He*, O2** 75, 75 -> 91 Se H2 78 Rb He 85 Sr He 88 Mo nogas 98 Ag nogas 107 Cd nogas 111 Sn nogas 118 Sb nogas 121 Te nogas 125 Cs he 133 Ba nogas 137 Gd nogas 157 Hg nogas 201 Tl nogas 205 Pb nogas 208 Bi nogas 209 U nogas 238
42 Experimental
For arsenic speciation analysis, aliquots of the samples were extracted with ultrapure water. The ratio sample : water was chosen depending on the total arsenic concentration and the available amount of sample. Typically, around 50 – 250 mg of sample were used (weighed to 0.1 mg), and at least 20 times more water was added. The mixtures were thoroughly shaken and then sonicated in an ultrasonic bath at room temperature for 15 minutes. Afterwards, they were centrifuged at 5500*g and filtered through 0.2 µm polyamide syringe filters (Marcherey-Nagel, Düren, Germany).
Usually, extractions were carried out in triplicates. In case of Cyanoboletus pulverulentus , each sample was only extracted once, because of the large number of samples from the same species. Extracts of samples with extremely high total arsenic concentrations were further diluted either 1+9 or 1+99 to a final expected concentration of roughly 100 µg As/L. Usually, one undiluted extract of each sample was injected at the end of the measurement to look for lower concentrated arsenic species.
Hydrogen peroxide was added to one replicate per sample (10 µL H 2O2 to 90 µL extract, directly in a 300 µL PP HPLC vial, closed with a snap cap). The vial was shaken on a vortex mixer and then put into a drying oven at 45°C for about 1 hour. Alternatively, if the oven was unavailable, the samples were put onto the warm surface of a running vacuum pump for about 1 hour. Through the H 2O2 and the elevated temperature, any labile compounds were oxidized to a much more stable form. For example, trivalent arsenicals would oxidize to their pentavalent equivalent, and thio-arsenicals would exchange the sulfur with an oxygen ion. These oxidized extracts were measured in addition to the untreated samples to get more information about the individual unidentified arsenic species and also served as a quality measure for the stability of the extracts and hence the validity of the results.
Extraction efficiencies were determined by diluting aliquots of the extracts with ultrapure water and nitric acid (10 % v/v) and then measuring the total arsenic concentrations similar to the digested samples with ICPMS.
Arsenic speciation analysis was carried out with HPLC (Agilent 1200, Agilent Technologies, Waldbronn, Germany) coupled to ICPQQQMS. All samples were measured with anion-exchange and cation- exchange chromatography. Both methods were validated [186]. The settings are listed in Table 7. The chemicals that are given for the mobile phases have shown to have a very low arsenic background and are therefore well suited for arsenic speciation analysis at trace concentrations.
43 Experimental
Table 7. Settings for ion-exchange chromatography. Feature Anion-exchange Cation-exchange PRP-X100 Zorbax 300-SCX Column name (Hamilton, Bonaduz, Switzerland) (Agilent, Waldbronn, Germany) 150*4.6 mm, 5 µm or Column dimensions 150*4.6 mm, 5 µm 250*2.1 mm, 5 µm 20 mM ammonium phosphate in Mobile phase 10 mM pyridine in water, pH 2.3 water, pH 6.0 Pyridine Ammonium dihydrogen phosphate Chemical for (Chromasolv® Plus, for HPLC, (99.99 %, Suprapur®, Merck KGaA, mobile phase ≥ 99.9 %, Sigma-Aldrich, Steinheim, Darmstadt, Germany) Germany) Chemical for Ammonia Nitric acid (sub-boiled) or formic acid adjusting pH (25 %, Suprapur®, Merck KGaA) (98-100 %, EMSURE®, Merck KGaA) 40 (newer columns have 30 as Temperature [°C] 30 limit, which also works well) 1 (150*4.6 mm) or Flow rate [mL/min] 1.5 0.4 (250*2.1 mm) Typical injection 20 (150*4.6 mm) or 20 volume [µL] 10 (250*2.1 mm)
The arsenic signal was recorded in oxygen reaction mode at m/z 75 91. Carbon dioxide (5 % in Argon, 15 % of the carrier gas flow) was added online between spray chamber and torch to enhance the arsenic signal and compensate possible differences in the carbon loads of the samples and the calibrations standards [172]. For quantification and identification, calibrations standards from 0.05 to 100 µg As L -1 were prepared from pure solutions of the different arsenic species. Arsenate, DMA and MA (and AB as a model compound for cationic arsenicals) were separated via anion-exchange chromatography (see a standard chromatogram in Figure 25A), while AB, TMAO, AC and TETRA were quantified with cation-exchange chromatography (Figure 26A). Several other pure arsenic compounds were only injected once per batch to compare their retention times with unknown arsenicals in the mushroom extract (see chromatograms in Figure 25B and Figure 26B and C). These compounds were: DMAA, DMAE; DMAP, DMAB, TMAP and four arsenosugars (glycerol, phosphate, sulfonate and sulfate; purified from seaweed). During the study on Elaphomyces spp. for publication 2, a pure solution of MA(III) was also injected and used for spiking experiments. This particular standard was kept frozen when not needed, to slow down oxidation to MA. Around one year after the preparation (dissolving
MeAsI 2 in water and 5 % methanol), about half of the MA(III) was converted to MA.
44 Experimental
Figure 25. Typical anion-exchange chromatograms of standard solutions. A: Standards used for quantification, each at 10 µg As/L. B: Other small arsenicals (10 µg As/L for each species, except DMAE (7 µg As/L) and sugars: gly = glycerol sugar, 7 µg As/L, phos = phosphate sugar, around 3 µg As/L). C: Long retained sulfonate (around 10 µg As/L) and sulfate (around 20 µg As/L) sugars.
45 Experimental
Figure 26. Typical cation-exchange chromatograms of standard solutions. A: Standards used for quantification, each at 10 µg As/L. B: Other small arsenicals (10 µg As/L for each species, except DMAE (7 µgAs/L) and sugars: gly = glycerol sugar, 7 µg As/L, phos = phosphate sugar, around 3 µg As/L).
In the publications 2 and 3, HPLC-ES-MS (6120, Agilent Technologies) was applied in addition to HPLC- ICPMS. In both cases, the same chromatographic method was used, which was optimized beforehand with HPLC-ICPMS: As already for the first cation-exchange method with HPLC-ICPMS, a Zorbax 300-SCX column was used. The mobile phase consisted of 0.5 M formic acid (98-100 %, EMSURE®, Merck KGaA) and 0.03 M ammonium formate (pH = 2.3) in channel A, and methanol in channel B. The ratio of the channels was 92:8 (A:B). The flow rate was 1.5 mL/min, but the flow was split after the column via a T- piece to reduce the input to the ES-MS (the rest was going to the waste). A chromatogram with ICPMS as detector is shown in Figure 27. Capillary voltage and fragmentor voltage of the ES-MS were optimized before the measurements (1000 and 90 V, respectively). The gas temperature was 350°C and the drying gas flow was 12 L/min.
46 Experimental
Figure 27. HPLC-ICPMS cation-exchange chromatogram of AB, TMAO, AC and TETRA with 0.5 M formic acid and 8 % methanol as mobile phase compatible with ES-MS.
In publication 2 ( Elaphomyces spp.) the ES-MS was used to confirm the identity of TMAO in the extracts at m/z 137 (MH +). In publication 3, ES-MS played a much more important role. In the chromatograms obtained by cation-exchange HPLC-ICPMS, a stable unknown arsenical (named UNK A) was detected eluting after TETRA. Extracts were then injected multiple times onto the cation-exchange column, and the effluent was collected around the retention time of UNK A. For this purpose, a column that had already been used many times was selected, because the retention times were much lower than with new columns, which enabled many more injections in the same time.
After concentrating it by freeze-drying, the presence and concentration of UNK A was controlled with HPLC-ICPMS. It turned out that only UNK A and TETRA were present in the solution, both at around 50 µg As/L. In the next step, the molecular mass of UNK A was investigated with ES-MS. The molecular mass of TETRA (m/z 135 for M +) was recorded as a time reference for UNK A. For UNK A, the mass range 70-500 was scanned. At the expected retention time of UNK A, a peak with m/z 179 was detected, which corresponds to the molecular mass of the hypothetical molecule homoarsenocholine. The exact mass for this compound was calculated for experiments with HPLC-high resolution ES-MS (Q-Exactive Hybrid Quadrupole-Orbitrap MS, Thermo Fisher Sci., Erlangen, Germany). The chromatographic setup was the same as for normal ES-MS. Again, the flow was split after the chromatographic column. With the Orbitrap MS, the exact mass 179.0411 was recorded, together with the exact masses of TMAO and TETRA. Standards of these two arsenicals were injected as retention time references. Further, MS/MS fragmentation experiments were carried out to see the characteristic fragments of the hypothesized homoarsenocholine. The settings of the Orbitrap measurement are given in Table S3 of publication 3.
47 Publications
4 Publications
4.1 Publication 1: Arsenic hyperaccumulation and speciation in the edible ink stain bolete ( Cyanoboletus pulverulentus )
48 Food Chemistry 242 (2018) 225–231
Contents lists available at ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Arsenic hyperaccumulation and speciation in the edible ink stain bolete (Cyanoboletus pulverulentus )
Simone Braeuer a, Walter Goessler a, Jan Kameník b, Tereza Konvalinková c, Anna Žigová d, Jan Borovi čka b,d,⁎ a University of Graz, Institute of Chemistry, Universitätsplatz 1, 8010 Graz, Austria b Nuclear Physics Institute, The Czech Academy of Sciences, Hlavní 130, 25068 Husinec- Řež, Czech Republic c Institute of Microbiology, The Czech Academy of Sciences, Víde ňská 1083, 14220 Prague 4, Czech Republic d Institute of Geology, The Czech Academy of Sciences, Rozvojová 269, 16500 Prague 6, Czech Republic
ARTICLE INFO ABSTRACT
This paper is dedicated to Tjakko Stijve on the The edible ink stain bolete ( Cyanoboletus pulverulentus ) was found to hyperaccumulate arsenic. We analyzed 39 occasion of his 80th birthday. individual collections determined as C. pulverulentus , mostly from the Czech Republic. According to our results, −1 Keywords: concentrations of arsenic in C. pulverulentus fruit-bodies may reach 1300 mg kg dry weight. In most collec- Edible mushrooms tions, data for total and bioavailable arsenic in underlying soils were collected but no signi ficant correlation Dimethylarsinic acid between the soil arsenic content and arsenic concentrations in the associated fruit-bodies was found. Within the Soil fruit-bodies, we found the majority of arsenic accumulated in the hymenium. Besides occasional traces of me- Health risk thylarsonic acid (MA), the arsenic speciation in all mushroom samples consisted solely of dimethylarsinic acid HPLC-ICPMS (DMA) and no inorganic arsenic was detected. Because of the carcinogenic potential of DMA, C. pulverulentus should not be recommended as an edible mushroom and its consumption should be restricted.
1. Introduction in varying proportions ( Nearing et al., 2014; Šlejkovec, Byrne, Stijve, Goessler, & Irgolic, 1997 ). The ink stain bolete, Cyanoboletus pulverulentus (Opat.) Gelardi, Since our former results for arsenic levels in several Czech collec- Vizzini & Simonini is a mushroom species of the family Boletaceae tions of C. pulverulentus reached hundreds of mg kg −1, which might growing in ectomycorrhizal symbiosis with various deciduous trees represent a possible hazard for consumers, we decided i) to study a (Fig. 1 A). Despite not being as common as more favored edible species representative number of collections from various sites, in order to like Imleria badia or Boletus edulis , it is frequently found in synanthropic inspect the variation of the total arsenic concentrations in this fungus sites (parks, gardens, avenues) and picked by mushroom hunters be- and distribution of arsenic in the fruit-body, ii) to determine the in- cause it has been recommended over decades as a good edible fungus in dividual molecular species of arsenic, in order to evaluate a possible the Czech Republic ( Smotlacha & Vejrych, 1947 ). Despite the fact that risk for consumers, and iii) to study the fungus –soil relationship and Cocchi and Vescovi (1996) pointed to highly elevated levels of arsenic calculate the bioaccumulation factor. in Italian collections, the phenomenon of arsenic accumulation in this fungus has never been investigated. 2. Materials and methods Arsenic can be highly accumulated in mushrooms, with levels commonly reaching tens but rarely also thousands of 2.1. Mushroom sampling and processing mg kg −1 dry weight ( Falandysz & Rizal, 2016 ). However, total arsenic concentrations provide no information about possible risk for con- We analyzed 39 individual collections determined as C. pulverulentus sumers. For example, the major arsenic species usually found in from the Czech Republic (36), France (1), Portugal (1), and the USA (1). mushrooms of the family Agaricaceae – arsenobetaine ( Nearing, Most of the samples were harvested in 2016. Mushrooms were collected Koch, & Reimer, 2014, 2016 ) – is considered non-toxic ( Kaise, by J. Borovi čka or donated by our co-workers. The distribution of the Watanabe, & Itoh, 1985; Newcombe et al., 2010 ). Besides arsenobe- sampling sites in the Czech Republic is indicated in Fig. 1 B (for the list taine, other arsenic species have also been found in various macrofungi, of all collections see Supplementary Table T1 ). Fruit-bodies were
⁎ Corresponding author at: Nuclear Physics Institute, The Czech Academy of Sciences, Hlavní 130, 25068 Husinec- Řež, Czech Republic. E-mail address: [email protected] (J. Borovi čka). http://dx.doi.org/10.1016/j.foodchem.2017.09.038 Received 29 June 2017; Received in revised form 31 August 2017; Accepted 6 September 2017
Available online 08 September 2017
0308-8146/ © 2017 Elsevier Ltd. All rights reserved. S. Braeuer et al. Food Chemistry 242 (2018) 225–231
Fig. 1. (A) Cyanoboletus pulverulentus , fruit-bodies of the collection CBP-15 (PRM 944013, EMBL-Bank LT714707). (B) Distribution of sampling sites in the Czech Republic. cleaned from substrate debris, either lyophilized or dried at 50 °C to peroxide was added to an aliquot of 15 exemplary extracts to a final constant weight, and pulverized in a grinder. In order to document the concentration of 10% v/v . The mixtures were shaken and put in an oven collections, herbarium specimens were deposited at the herbarium of for one hour at 45 °C. The pure extracts as well as the extracts mixed the Mycological Department, National Museum, Prague (PRM) or the with hydrogen peroxide were further diluted 1:10 with ultrapure water Museum of Eastern Bohemia in Hradec Králové (HR). (samples with a total arsenic concentration up to 200 mg kg −1) or even In selected collections, ITS rDNA (ITS1, 5.8S, and ITS2 sequences) 1:100 (samples with a total arsenic concentration higher than and the D1-D2 domain at the 5 ′ end of the nuclear LSU rRNA gene were 200 mg kg −1), to avoid an overload of the analytical column. The di- sequenced. DNA was extracted according to Janda, K říž, Konvalinková, luted samples were subjected to high-performance liquid chromato- and Borovi čka (2017) ; for further details (PCR conditions, amplicon graphy (HPLC 1200, Agilent Technologies) coupled to an ICPQQQMS. puri fication, etc.) see our former studies ( Borovi čka, Noordeloos, Dimethylarsinic acid (DMA), methylarsonic acid (MA), and arsenate (As Gryndler, & Oborník, 2011; Borovi čka et al., 2015 ). Sequences were (V)) were separated via anion-exchange chromatography, while ar- submitted to EMBL-Bank and GenBank under the accession numbers senobetaine (AB), trimethylarsine oxide (TMAO), arsenocholine (AC), LT714704 –LT714709 and MF373585, respectively. LSU rRNA gene and the tetramethylarsonium ion (TETRA) were separated via cation- sequences (MF373585 sequence and appropriate sequences down- exchange chromatography. For anion-exchange chromatography, a loaded from the GenBank database) were aligned with MAFFT online PRP-X100 column (150 × 4.6 mm, 5 µm; Hamilton, Bonaduz, version 7 ( Katoh & Toh, 2008 ) with default settings. The evolutionary Switzerland) was used as stationary phase, and the mobile phase was an history was inferred by using the Maximum Likelihood method based aqueous solution of 20 mM ammonium phosphate, pH 6.0. Cation-ex- on the Tamura-Nei model in MEGA7 ( Kumar, Stecher, & Tamura, 2016; change chromatography was carried out with a Zorbax 300-SCX column Tamura & Nei, 1993 ). (150 × 4.6 mm, 5 µm; Agilent, Waldbronn, Germany) and a 10 mM aqueous pyridine bu ffer, pH 2.3. Both methods are described in more 2.2. Analyses of mushrooms detail by Scheer et al. (2012) . Chromatograms are shown in Supplementary Fig. F1 . The arsenic signal was recorded again in oxygen All samples were digested in triplicate with nitric acid (p.a., ≥65%; reaction mode at m/z 75 → 91. Carbon dioxide (1% in argon) was Carl Roth, Karlsruhe, Germany) in a microwave-assisted autoclave at added online between spray chamber and torch to enhance the arsenic ff 250 °C for 30 min. The digests were diluted with ultrapure water signal and compensate for possible di erences in the carbon loads of (18.2 M cm; Merck Millipore, Darmstadt, Germany) to a final acidity the samples and the calibrations standards ( Kova čevi č & Goessler, Ω fi fi of 10% v/v . The total arsenic concentrations were determined with an 2005 ). For quanti cation and identi cation, calibration standards from −1 inductively coupled plasma triple quadrupole mass spectrometer 0.05 to 100 µg As L were prepared from pure solutions of the dif- (ICPQQQMS; Agilent 8800, Agilent Technologies, Waldbronn, ferent arsenic species. ffi Germany; settings and performance are listed in Supplementary Table To obtain information about the extraction e ciency, aliquots of T2 ). Arsenic was measured in oxygen reaction mode at m/z 75 → 91. the extracts were diluted with ultrapure water and nitric acid (10% v/ Germanium was used as internal standard. The certi fied standard re- v). The total arsenic concentrations were determined as already de- ference material SRM 1573a (Tomato Leaves; NIST, Gaithersburg, MD, scribed for the digested samples. USA) was used for quality control. Variations of arsenic concentrations in fruit-bodies of various sizes 2.3. Soil sampling and analysis collected at a single sampling site were tested in 16 samples (CBP 11a –p). In these samples, arsenic was determined by instrumental In order to document the environment and to investigate the fun- neutron activation analysis (INAA) according to Borovi čka, Řanda, and gus –soil relationship, soils were collected as a representative composite Jelínek (2005) . Data on total arsenic distribution within the fruit-bodies of the organomineral Ah horizon at 34 sites. Soils were dried at room from two individual sampling sites (samples CBP-10b and CBP-25b) are temperature and sieved through a 2-mm stainless steel mesh. The pH also based on INAA results. Certi fied standard reference material SRM values were potentiometrically measured ( van Reeuwijk, 2002 ) in dis- 1633b (Coal Fly Ash; NIST, Gaithersburg, MD, USA) was used as con- tilled water and in 1 M KCl with a SenTix electrode using a soil:solution trol. Possible associations between variables were tested by use of non- ratio of 1:2.5. The pH values were assessed by the scale of Baize (1993, parametric Spearman ’s Rho test and results at p < 0.05 were con- chap. 10) . A representative part of each sample was milled in an agate sidered statistically signi ficant. mill (Fritsch, Germany) and aliquots of approximately 250 mg were For arsenic speciation analysis, about 50 mg (weighed to 0.1 mg) used for determination of total element contents by INAA with epi- were extracted with 2 mL of ultrapure water, sonicated in an ultrasonic thermal neutrons ( Cejpková et al., 2016 ). Certi fied standard reference bath for 15 min, centrifuged at 5500 g and then filtered through 0.2 µm material SRM 2711a (Montana II Soil; NIST, Gaithersburg, MD, USA) Nylon ® syringe filters (Macherey-Nagel, Düren, Germany). Hydrogen was used as control.
226 S. Braeuer et al. Food Chemistry 242 (2018) 225–231
Furthermore, we sequentially analyzed two arsenic soil fractions concentrations in soils ( Table 2 ) found at all investigated sites varied (“non-speci fically sorbed ” and “speci fically adsorbed ”), which are from 6.1 to 36.2 mg kg −1 (13.9 ± 6.6 mg kg −1 As on average). The considered the most bioavailable ( Wenzel et al., 2001 ). In 50-mL cen- sum of the “non-speci fically sorbed ” and “speci fically adsorbed ” arsenic trifuge tubes, 1.600 ± 0.005 g of milled soil with 40 mL added 0.05 M fractions, i.e. the most bioavailable soil arsenic, represented circa fi (NH 4)2SO 4 (Suprapur, Merck) were shaken for 4 h in an overhead 3–12% of total arsenic and there was a signi cant positive correlation shaker (Reax 2; Heidolph, Schwabach, Germany) at room temperature. between bioavailable and total arsenic in soil. No certi fied reference After centrifugation, the supernatant liquid was carefully decanted, material is available for quality assurance of the sequential arsenic filtered (0.45 µm ProFill PTFE filters, Fisherbrand) and analyzed by extraction by Wenzel et al. (2001) ; our results obtained for BCR-483 are inductively coupled plasma sector field mass spectrometer (ICPSFMS, included in Table 2 (presented for the first time). fi ff Element2, Thermo Scienti c). Then, 40 mL of 0.05 M NH 4H2PO 4 At all investigated sites, arsenic was e ectively accumulated in (TraceSELECT, Fluka) were added to the residue and samples were C. pulverulentus , with BAF tot 3–102 and BAF mob 39 –1095. However, shaken for 16 h and further processed and analyzed as described above. BAFs largely varied even among fruit-bodies at particular sites, which is Unmilled certi fied reference material BCR-483 (Sewage Sludge apparent from Fig. 2 . There was no signi ficant correlation between Amended Soil, IRMM) and procedural blanks were processed in the arsenic in fruit-bodies and total or bioavailable arsenic in soil. same way. In order to evaluate the arsenic uptake by the fungus, the bioac- 3.3. Arsenic speciation cumulation factor (BAF; Falandysz & Borovi čka, 2013 ) was calculated for sites where soils were collected. BAF values are related to both total Around 80% of the total arsenic was extractable with pure water ffi arsenic (BAF tot ) and bioavailable arsenic soil fraction (BAF mob ), i.e. to (see Table 1 ). The lowest extraction e ciency, 42%, was observed for the sum of “non-speci fically sorbed ” and “speci fically adsorbed ” arsenic the sample with the lowest total arsenic concentration (2.4 mg kg −1). fractions. There was only one other sample where less than 75% of the total ar- senic was extractable (CBP-03, 65%, 120 mg kg −1 total As). Of the 3. Results extracted arsenic, between 78 and 110% was detectable and quanti fi- able as arsenic species with HPLC-ICPMS, which are acceptable column 3.1. Total arsenic in mushrooms recoveries for natural biological samples. Overall, 43 –110% (mean: 71 ± 9%) of the total arsenic was quanti fied via speciation analysis. The trueness of the measured arsenic concentration in the certi fied DMA accounted for more than 99% of the detected arsenic species in all reference material SRM 1573a was very good, namely 106 ± 15% extracts. In 31 samples, no other arsenic compound was detected be- (n = 8). The single INAA result for SRM 1633b was sides DMA (example anion- and cation-exchange chromatograms are 126.0 ± 2.5 mg kg −1 (mass fraction ± combined uncertainty) which displayed in Supplementary Fig. F1 ), with a limit of detection of about is somewhat lower than the certi fied concentration range 0.02 mg kg −1 for each compound. The remaining 8 samples contained (136.2 ± 2.6 mg kg −1) but still within 10% of the certi fied con- traces of MA, namely 0.04 –0.52 mg kg −1, which is less than 0.2% of the centration range. Arsenic concentrations (all concentrations are ex- total arsenic. The addition of hydrogen peroxide did not cause any pressed on a dry weight basis) found in fruit-bodies of C. pulverulentus changes of the arsenic speciation in the extracts. varied across a large range of 2.4 –1300 mg kg −1 (see summary in Table 1 and results for the individual samples in Supplementary Table 4. Discussion T3 ). The mean arsenic concentration was 250 ± 260 mg kg −1, and the median was a bit lower at 160 mg kg −1. No signi ficant correlation Arsenic concentrations in soils found at all sites fall within the range between arsenic concentration and fruit-body dry weight was observed of common arsenic concentrations (compare with Kabata-Pendias, (Fig. 2 A) but the total amount of arsenic in particular fruit-bodies was 2011, chap. 20 and Borovi čka et al., 2017 ) and the accumulation of signi ficantly positively correlated with fruit-body dry weight ( Fig. 2 B). arsenic in C. pulverulentus thus represents a natural biological phe- The arsenic distribution within the fruit-body of C. pulverulentus was nomenon not related to elevated arsenic content in the soil environ- investigated in two specimens from distinct sites. The results ( Fig. 3 ) ment. The term ‘hyperaccumulator ’ is used for species containing at showed that arsenic was highly accumulated in the hymenium (tubes). least 100 times higher concentrations of a particular element than other In both tested collections, circa 80% of total arsenic was in the hyme- species growing over underlying substrate of the same characteristics nium and much lower contents were observed in the cap flesh (Brooks, 1998 ). As common arsenic concentrations in ectomycorrhizal (11 –16%) and stipe (3 –9%). fungi from pristine areas only rarely exceed 10 mg kg −1 (Falandysz & Rizal, 2016; Vetter, 2004 ), C. pulverulentus can be con- 3.2. Soil characteristics sidered an arsenic hyperaccumulator, similarly to the ascomycete Sar- cosphaera coronaria , which also accumulates this element at levels ex- Apparently, C. pulverulentus occurs in very acid to neutral soils; the ceeding 1000 mg kg −1 (Byrne et al., 1995 ). Another boletoid species, median pH value corresponds to a weakly acid reaction. The analyzed Imleria badia , can also reach high arsenic concentrations in its fruit- arsenic concentration in SRM 2711a (102.0 ± 2.1 mg kg −1) fell within bodies (up to 490 mg kg −1) but such samples have been reported only the certi fied range (NIST value 107 ± 5 mg kg −1). Arsenic from extremely As-polluted environments ( Mleczek et al., 2016;
Table 1 Arsenic concentration, extraction e fficiency (extracted/total As) and As species in Cyanoboletus samples ( n = 39); *MA: n = 8, not detected in the other 31 samples. **Missing As = total As − (DMA + MA).
Total As Extraction e fficiency Column recovery DMA MA * Missing As ** [mg kg −1] [%] [%] [% of extracted As] [% of extracted As] [% of total As]
Mean 250 83 85 85 0.10 30 Standard deviation 260 9 6 6 0.04 8 Median 160 84 84 84 0.09 29 Range 2.4 –1300 42 –99 78 –110 79 –110 0.04 –0.19 0 –58
227 S. Braeuer et al. Food Chemistry 242 (2018) 225–231
Fig. 2. (A) Relationship between As concentration (mg kg −1) and fruit-body dry weight (g) in Cyanoboletus pulverulentus , collection CBP-11 ( n = 16); linear regression is indicated. (B) Relationship between total As content (µg) in fruit-bodies and fruit-body dry weight (g) in the same collection; linear regression is indicated.
Niedzielski, Mleczek, Magdziak, Siwulski, & Kozak, 2013 ). in the rest of the cap and the lowest in the stipe. We observed the same However, accumulation of arsenic by macrofungi is a poorly un- distribution in two samples of C. pulverulentus (Fig. 3 ), although we did derstood phenomenon. As demonstrated in Fig. 2 B, total arsenic was not analyze the spores. signi ficantly positively correlated with fruit-body dry weight. It can be Our results showed an extremely large range of arsenic concentra- assumed that fruit-body dry weight more or less corresponds to the tions in the investigated fruit-bodies. The two lowest arsenic con- fruit-body age. It appears that the flux of arsenic from the mycelium to centrations were found in the samples from Madeira Island (Portugal) the fruit-body is a continuous process; this was also observed in Lyco- and from the USA. With 3.2 and 2.4 mg kg −1, they contained around 10 perdon perlatum (Borovi čka et al., 2010 ), which is known to contain times less arsenic than the lowest of the rest of the samples mainly AB ( Šlejkovec et al., 1997 ). According to the review by Kala č (27 mg kg −1). We therefore sequenced the Cyanoboletus collections (2010) , most of the elements accumulated in mushrooms are dis- from Madeira and the USA along with 5 Czech samples with various tributed unevenly within a fruit-body; the highest contents are usually arsenic concentrations ( Supplementary Table T1 ), in order to recognize observed in the spore-forming part of the cap (but not in spores), lower a possible occurrence of more than one biological species in the dataset. The comparison of the ITS rDNA sequences using the BLAST searching tool at https://blast.ncbi.nlm.nih.gov/Blast.cgi revealed that the American collection (ASP-82, EMBL-Bank sequence LT714710, Mush- room Observer identi fier 245588) is signi ficantly di fferent from the European (85.5% similarity) and thus does not represent the genuine C. pulverulentus but another species (possibly undescribed). The phylo- genetic analysis (LSU rRNA) clearly showed its a ffinity to the genus Cyanoboletus (Supplementary Fig. F2 ). However, all other sequenced collections (ITS rDNA) from both continental Europe and Madeira Is- land were identical, which does not indicate the existence of more than one biological species. According to this fact, the large arsenic con- centration range observed in C. pulverulentus cannot be explained by the possible existence of accumulating and non-accumulating Cyanoboletus species in Europe. However, a large variation of element concentrations in fruit-bodies has also been reported for other hyperaccumulators, e.g., Amanita species ( Bene š, Hlo žková, Mat ěnová, Borovi čka, & Kotrba, 2016; Gryndler, Hr šelová, Soukupová, & Borovi čka, 2012 ); the factors in flu- encing such di fferences are poorly understood. Arsenic soil content and its bioavailability might, at least to some extent, in fluence arsenic ac- cumulation by C. pulverulentus . But a more than a tenfold di fference can be seen in arsenic concentrations in fruit-bodies collected from soils with similar characteristics (compare, e.g., CBP-18 and CBP-29, Supplementary Table T3 ). Furthermore, arsenic was not signi ficantly correlated with total arsenic in soils or the percentage of bioavailable arsenic. Our data thus do not indicate a major role of total arsenic soil content and fractionation in fungal accumulation, which is a phenom- enon recently observed by Kubrová et al. (2014) , who investigated accumulation of metals in macrofungi by using the BCR soil metal se- quential extraction procedure. Cyanoboletus pulverulentus is an ectomycorrhizal fungus and the Fig. 3. Distribution of arsenic concentrations ( figures indicated in fruit-bodies, mg kg −1) possibility that host plants play a role in arsenic accumulation should and total arsenic contents (µg) and their percentage in two individual fruit-bodies of Cyanoboletus pulverulentus : collection CBP-10b (fruit-body dry weight 6.23 g) and col- also be considered. We have sampled collections associated with var- lection CBP-25b (fruit-body dry weight 4.94 g). Arsenic concentrations in complete fruit- ious trees ( Supplementary Table T1 ) but no apparent relationship be- bodies and underlying soils (total and bioavailable arsenic: sum of both extracted frac- tween arsenic accumulation and particular tree host could be seen. In tions) are also indicated. mixed forest stands, it is di fficult to determine the association with a
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Table 2 Data on soils collected at particular sites of Cyanoboletus pulverulentus : pH values, total arsenic (As), and mobile fractions of arsenic (As F1 – sulfate extraction, As F2 – phosphate extraction, As ∑F: sum of fractions F1 and F2). Bioaccumulation factors are calculated for particular collections of C. pulverulentus and related to the total As soil concentration (BAF TOT ) and mobile As in soil (BAF MOB ).
−1 −1 −1 −1 Sample pH (H 2O) pH (KCl) As [mg kg ] As (F1) [µg kg ] As (F2) [µg kg ] As ( ∑F) [µg kg ] BAF TOT BAF MOB
CBP-01 5.01 4.14 25.6 112 1389 1501 10 177 CBP-02 4.22 3.45 26.6 172 1674 1846 15 217 CBP-03 4.58 3.66 12.6 109 545 653 9 180 CBP-04 3.89 3.24 17.5 82.0 903 985 14 251 CBP-05 6.21 5.47 13.5 106 593 699 3 64 CBP-06 4.65 3.73 36.2 65.2 1149 1214 3 90 CBP-07 5.27 4.37 15.9 60.0 1170 1230 9 123 CBP-08 5.84 5.13 8.45 58.7 272 331 10 246 CBP-09 4.41 3.68 21.3 184 1777 1961 4 39 CBP-10 4.92 3.96 9.78 29.5 359 389 16 412 CBP-11 5.54 4.68 12.6 41.6 463 505 4 –30 106 –759 CBP-12 5.56 4.81 8.24 83.5 474 558 15 225 CBP-13 3.87 3.23 12.1 94.1 689 783 22 347 CBP-14 6.53 6.2 18.1 106 2032 2138 6 49 CBP-15 6.3 5.75 8.16 53.5 508 561 16 –28 234 –402 CBP-16 5.84 5.12 12.3 38.6 672 711 18 312 CBP-17 6.62 6.1 18.3 103 1241 1345 36 493 CBP-18 5.32 4.44 7.96 41.3 588 630 6 77 CBP-19 4.64 3.75 6.83 26.6 281 307 4 87 CBP-20 4.43 3.73 11.9 189 694 883 21 281 CBP-21 6.36 5.65 20.7 116 766 882 17 394 CBP-22 4.27 3.3 9.36 30.2 480 510 53 978 CBP-23 4.45 3.84 10.8 33.8 779 812 48 643 CBP-25 5.28 4.32 10.1 39.5 313 352 7 210 CBP-26 5.85 4.96 15.0 38.4 811 850 17 308 CBP-27 7.05 6.79 7.88 163 443 606 77 1007 CBP-28 5.36 4.79 13.9 117 1201 1318 94 988 CBP-29 4.59 3.86 7.86 171 565 735 102 1095 CBP-30 5.57 4.91 8.80 82.6 374 457 45 857 CBP-31 4.86 3.92 8.94 34.6 384 418 26 551 CBP-33 3.63 3.05 19.1 74.1 1151 1225 4 58 CBP-34 3.83 3.12 6.05 43.8 390 434 12 163 CBP-35 3.88 3.18 9.64 71.5 572 643 5 79 ASP-36 5.24 4.54 21.9 88.2 1178 1266 14 248 BCR-483 – – – 1354 3331 4685 – – SRM-2711a – – 102 – – – –– particular tree species. Both low and high arsenic concentrations in and then detected with our analysis method. On the other hand, it could fruit-bodies (compare the collections CBP-17 and CBP-18) were ob- be possible that small arsenic species are strongly attached to bigger served in pure Tilia stands. However, high arsenic concentrations have molecules within the fruit-body, but proof for this hypothesis is missing. also been detected in collections from sites with all other potential host Of course, an optimization of the extraction method, for example by trees. including a second extraction step or extending the extraction time, The arsenic speciation of C. pulverulentus is highly interesting. could probably enhance the extraction e fficiency. It would be very in- Without exception, all 39 samples contained almost exclusively DMA. teresting to improve the column recovery as well. Unfortunately, both No inorganic (As (V) and As (III)) or cationic arsenic species were de- tasks were beyond the scope of our study and have to remain for future tectable with our method. The only other arsenic compound that was investigations. present in 8 samples was MA, but it accounted only for 0.04 –0.2% of Hyperaccumulation of arsenic in C. pulverulentus raises a serious the extracted arsenic. There was no di fference in the arsenic speciation question on the mushroom ’s edibility. DMA is considered to be much between samples from di fferent origins or soil arsenic concentration. less toxic than inorganic arsenic, at least concerning the acute toxicity. Especially the complete absence of inorganic arsenic is surprising be- However, DMA is a probable human carcinogen according to the United cause this is usually the dominating arsenic form in soil extracts ( Sun, States Environmental Protection Agency (US EPA) and de finitely car- Ma, Yang, Lee, & Wang, 2015 ). It has to be noted that this is not the first cinogenic according to the International Agency for Research on Cancer report of a mushroom that contains almost only DMA. For example, in a (IARC) ( IARC, 2012; US EPA, 2016 ). Therefore, the US EPA derived a publication from 1998 DMA was identi fied as the main arsenic species benchmark dose lower limit (BMDL10 – for a 10% increase in risk) of in the arsenic-accumulating mushroom Laccaria amethystina , besides 430 µg per kg body weight per day for DMA ( US EPA, 2006 ). According small amounts of MA, TMAO and As (V) ( Larsen, Hansen, & Gössler, to the European Food Safety Agency (EFSA), risk assessment of geno- 1998 ). toxic and carcinogenic substances should be carried out by calculating It is also noticeable that the detected DMA does not account for the margin of exposure (MoE) ( EFSA, 2005 ). The MoE is calculated by 100% of the total arsenic in our samples. Both extraction e fficiency and dividing the BMDL by the daily exposure. When using a BMDL10 that is column recovery were around 85% (see Supplementary Table T3 ). We derived from animal studies, an MoE of 10,000 or higher can be con- think that some of the arsenic that was not extracted could be DMA, sidered safe ( EFSA, 2005 ). because maybe the large amount of DMA was not extractable to 100% When calculating the MoE for an average person of about 70 kg in a single extraction step. We also think that it is not very likely that body weight and using the median DMA concentration in fungal bio- the missing arsenic is inorganic arsenic or a small, water-soluble or- mass (115 mg As kg −1 dry weight, which is roughly 12 mg As kg −1 ganic arsenical, because they would have been extractable with water fresh weight; N.B., concentrations only refer to the arsenic, not to the
229 S. Braeuer et al. Food Chemistry 242 (2018) 225–231
Table 3 Appendix A. Supplementary data Risk evaluation of consuming C. pulverulentus . BMDL10 = 430 µg kg −1 bw day −1. Average body weight of 70 kg is considered. Supplementary data associated with this article can be found, in the
DMA in C. DMA in C. Maximum Average daily Margin online version, at http://dx.doi.org/10.1016/j.foodchem.2017.09.038 . pulverulentus pulverulentus safe intake exposition of exposure References Selected [µg As kg −1 fresh [kg per year] [µg kg −1 bw (MoE) concentration mass] day −1] Baize, D. (1993). Soil science analyses. A guide to current use. Chichester: John level Wiley & Sons . Bene š, V., Hlo žková, K., Mat ěnová, M., Borovi čka, J., & Kotrba, P. (2016). Accumulation Median 12,000 0.090 0.042 10,173 of Ag and Cu in Amanita strobiliformis and characterization of its Cu and Ag uptake Lowest 100 10 0.039 10,987 transporter genes AsCTR2 and AsCTR3. Biometals, 29 , 249 –264. http://dx.doi.org/ Highest 86000 0.012 0.040 10,646 10.1007/s10534-016-9912-x . Borovi čka, J., Braeuer, S., Žigová, A., Gryndler, M., Dima, B., Goessler, W., ... Kärcher, R. (2017). Resurrection of Cortinarius coalescens : Taxonomy, chemistry, and ecology. 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231 Arsenic hyperaccumulation and speciation in the edible ink stain bolete ( Cyanoboletus pulverulentus )
Simone Braeuer a, Walter Goessler a, Jan Kameník b, Tereza Konvalinková c, Anna Žigová d, Jan Borovička b,d,*
aUniversity of Graz, Institute of Chemistry, Universitätsplatz 1, 8010 Graz, Austria bNuclear Physics Institute, The Czech Academy of Sciences, Hlavní 130, 25068 Husinec- Řež, Czech Republic cInstitute of Microbiology, The Czech Academy of Sciences, Vídeňská 1083, Prague 4, Czech Republic dInstitute of Geology, The Czech Academy of Sciences, Rozvojová 269, 16500 Prague 6, Czech Republic
*Corresponding author. E- mail address: [email protected] (J. Borovička). Tel.: +420 777 008 658.
SUPPORTING INFORMATION
Food Chemistry (2017)
Content: Supplementary Table T1 Supplementary Table T2 Supplementary Table T3 Supplementary Figure F1 Supplementary Figure F2
1 Supplementary Table T1. Analyzed Cyanoboletus collections with data on sampling sites, associated host plants, herbarium vouchers and molecular data (EMBL-Bank, GenBank).
molecular ID date country locality host plants at site herbarium data CBP-01 3.6.2016 Czech Rep. Prague, Petřín Hill Betula, Fagus PRM 944069 - CBP-02 9.6.2016 Czech Rep. Prague, Cibulka Park Quercus, Carpinus PRM 944014 LT714705 CBP-03 8.6.2016 Czech Rep. Hluboká n. Vlt. Fagus - - CBP-04 14.6.2016 Czech Rep. Poříčko n. Sáz. Carpinus, Picea PRM 857481 - CBP-05 11.6.2016 France Corrèze, region Limousin Tilia, Picea PRM 944020 - CBP-06 20.6.2016 Czech Rep. Vlašim Tilia, Quercus, Carpinus PRM 944011 - CBP-07 10.6.2016 Czech Rep. Čerčany (viaduct) Tilia, Quercus PRM 944001 LT714706 CBP-08 20.6.2016 Czech Rep. Čerčany Tilia - - CBP-09 21.6.2016 Czech Rep. Prague-Klánovice, Úvaly Tilia, Quercus PRM 944015 - CBP-10 16.6.2016 Czech Rep. Žebračka (Přerov) Tilia PRM 944006 - CBP-11 16.6.2016 Czech Rep. Žebračka (Přerov) Tilia - - CBP-12 22.6.2016 Czech Rep. Jablonné v Podještědí Tilia, Quercus, Carpinus PRM 944019 - CBP-13 22.6.2016 Czech Rep. Jablonné v Podještědí Tilia, Picea PRM 944018 - CBP-14 25.6.2016 Czech Rep. Rančice Tilia, Picea - - CBP-15 17.6.2016 Czech Rep. Jesenice (Rakovník) Quercus, Corylus PRM 944013 LT714707 CBP-16 25.6.2016 Czech Rep. Heřmanův Městec Carpinus PRM 944021 - CBP-17 19.6.2016 Czech Rep. Úsobí (Humpolec) Tilia PRM 944022 LT714708 CBP-18 8.6.2016 Czech Rep. Jindřichův Hradec (JH1) Tilia PRM 935997 LT714709 CBP-19 8.6.2016 Czech Rep. Jindřichův Hradec (JH2) Tilia - - CBP-20 15.6.2016 Czech Rep. Borovany Tilia, Quercus, Fagus PRM 944029 - CBP-21 8.6.2016 Czech Rep. Drachkov (Strakonice) Quercus - - CBP-22 3.7.2016 Czech Rep. Rychnov na Moravě Quercus, Corylus, Tilia HR 102057 - Picea, Ulmus, Fagus, CBP-23 10.8.2016 Czech Rep. Lichnice- Kaňkovy hory HR 99971 - Carpinus CBP-24 8.7.2016 Czech Rep. Rožmitál (Broumov) Picea, Fagus, Corylus HR 101207 - CBP-25 7.6.2016 Czech Rep. Vrchovnice Corylus HR 102048 - CBP-26 24.6.2016 Czech Rep. Bojiště (Ledeč n. Sázavou) Tilia, Quercus, Picea HR 102059 - CBP-27 17.8.2016 Czech Rep. Ústí n. Labem, Vaňov Betula - - CBP-28 24.7.2016 Czech Rep. Stvolínky Tilia - - CBP-29 6.8.2016 Czech Rep. Kersko Tilia, Quercus - - CBP-30 7.8.2016 Czech Rep. Prague, Homolka Pond Quercus - - CBP-31 25.6.2016 Czech Rep. Svitavy Tilia - - CBP-32 15.8.2016 Czech Rep. Lipník n. Bečvou Quercus, Carpinus, Fagus - - Žofínský prales (Pohorská CBP-33 2005 Czech Rep. Fagus - - Ves) CBP-34 26.9.2005 Czech Rep. Nadějov Hill (Staňkov) Fagus - - Chlum u Třeboně, Bukové CBP-35 27.9.2005 Czech Rep. Fagus - - kopce CBP-36 30.7.2016 Czech Rep. Jihlava, Heulos Park Tilia, Quercus, Fagus - - ASP-26 26.9.2015 Portugal Madeira, Ribeiro Frio Quercus PRM 935923 LT714704 ASP-36 6.9.2015 Czech Rep. Lázně Bohdaneč Tilia HR 90200 - ASP-82 23.7.2016 USA NY, Oneida County Fagus, Betula PRM 944518 LT714710, MF373585
2 Supplementary Table T2. (A) Instrument settings and performance of ICPQQQMS as used for total arsenic analysis. Settings for speciation analysis were similar; any deviating settings are given in brackets. (B) Instrument settings and performance of ICPSFMS as used for analysis of bioavailable soil arsenic.
A. ICPQQQMS settings: total arsenic analysis (speciation analysis) Scan Type MS/MS RF Power 1600 W RF Matching 1.8 V Smpl Depth 8 mm Carrier Gas 1.1 (0.85) L/min Option Gas 0 (15) % Nebulizer Pump 0.1 (0.5) rps S/C Temp 2 °C Extract 1 0 V Extract 2 -160 V Omega Bias -90 V Omega Lens 6.6 V Q1 Entrance -1 V Q1 Exit -1 V Cell Focus 2 V Cell Entrance -50 V Cell Exit -60 V Deflect 3.2 V Plate Bias -60 V Q1 Bias -2 V Q1 Prefilter Bias -44 V Q1 Postfilter Bias -180 V 4th cell gas flow 25 % OcP Bias -5 V OcP RF 200 V intensity (1 µg/L As, m/z 75 -> 91) ~12000 CPS
B. ICPSFMS Element 2 (analysis of bioavailable soil arsenic) Resolution mode LR/HR RF Power 1200 W Cool Gas 16 L/min Sample Gas 0.955 L/min Auxiliary Gas 0.96 L/min Peristaltic Pump 4.5 rpm Spray Chamber Type Quartz Double-Pass Lenses (V) Extraction -2000 V Focus -1165 V X-Deflection 6.9 V Y-Deflection 7.05 V
3 Shape 120 V High Resolution Lenses (V) Quad 1 2.41 V Quad 2 -2.51 V Focus Quad -7.15 V SEM Deflection -15 V SEM Voltage (V) 1910 V Sensitivity (1 ng/g As) Low Mass Resolution ~140000 CPS Sensitivity (1 ng/g As) High Mass Resolution ~1400 CPS
4 Supplementary Table T3 . Total arsenic and arsenic species concentrations (in dry mass) of the individual fruit-bodies of Cyanoboletus collections and total/bioavailable arsenic concentrations (in dry mass) in corresponding soils.
extracted column biovailable total As DMA MA total soil As sample As recovery soil As [mg kg -1 ] [mg kg -1 ] [%] [mg As kg -1 ] [mg As kg -1 ] [mg kg -1 ] [µg kg -1 ] ASP-26 3.2 ± 0.1 2.8 82 2.3 < 0.02 21.9 1266 ASP-36 314 ± 5 311 112 349 0.3 n.a. n.a. ASP-82 2.4 ± 0.1 1 100 1.0 < 0.02 n.a. n.a. CBP-01 270 ± 30 220 86 190 < 0.02 25.6 1501 CBP-02 400 ± 20 340 88 300 0.29 26.6 1846 CBP-03 120 ± 10 76 86 65 < 0.02 12.6 653 CBP-04 250 ± 30 210 86 180 < 0.02 17.5 985 CBP-05c 45 ± 4 43 86 37 0.08 13.5 699 CBP-06 109 ± 8 91 84 76 < 0.02 36.2 1214 CBP-07 150 ± 10 130 85 110 < 0.02 15.9 1230 CBP-08 81 ± 4 70 84 59 < 0.02 8.45 331 CBP-09f 76 ± 9 65 83 54 < 0.02 21.3 1961 CBP-10a 160 ± 3 140 84 118 < 0.02 9.78 389 CBP-11a 78 ± 4 63 84 53 < 0.02 12.6 505 CBP-12 130 ± 10 100 90 90 < 0.02 8.24 558 CBP-13 270 ± 20 220 82 180 < 0.02 12.1 783 CBP-14 105 ± 4 80 84 67 < 0.02 18.1 2138 CBP-15e 149 ± 3 130 85 110 < 0.02 8.16 561 CBP-16b 220 ± 10 190 84 160 < 0.02 12.3 711 CBP-17 660 ± 50 560 82 460 < 0.02 18.3 1345 CBP-18 49 ± 5 43 81 35 < 0.02 7.96 630 CBP-19 27 ± 2 23 87 20 < 0.02 6.83 307 CBP-20a 250 ± 30 200 85 170 < 0.02 11.9 883 CBP-21 350 ± 20 270 85 230 < 0.02 20.7 882 CBP-22 500 ± 40 430 84 360 < 0.02 9.36 510 CBP-23 520 ± 30 450 82 370 < 0.02 10.8 812 CBP-24 140 ± 4 110 91 100 < 0.02 n.a. n.a. CBP-25a 74 ± 7 66 82 54 < 0.02 10.1 352 CBP-26 262 ± 9 220 82 180 < 0.02 15.0 850 CBP-27 610 ± 100 470 83 390 0.52 7.88 606 CBP-28 1300 ± 60 1070 80 860 0.4 13.9 1318 CBP-29 810 ± 30 650 82 530 < 0.02 7.86 735 CBP-30 390 ± 20 350 80 280 0.34 8.80 457 CBP-31 230 ± 4 190 79 150 < 0.02 8.94 418 CBP-32 208 ± 7 180 78 140 < 0.02 n.a. n.a. CBP-33 71 ± 7 63 79 50 < 0.02 19.1 1225 CBP-34 71 ± 7 59 80 47 < 0.02 6.05 434 CBP-35 51 ± 4 44 86 38 0.04 9.64 643 CBP-36 160 ± 2 140 86 120 0.09 n.a. n.a.
5 Supplementary Figure F1 . (A) Anion-exchange chromatogram of and a calibration standard containing 10 µg As L-1 of AB, DMA, MA and As (V). (B) Detail of the extract’s anion -exchange chromatogram. (C) Cation-exchange chromatogram of an extract (CBP-21) a calibration standard containing 10 µg As L-1 of AB, TMAO, AC, and TETRA.
6 Supplementary Figure F2. Evolutionary analysis (LSU rRNA) by Maximum Likelihood method showing the phylogenetic placement of the unidentified American collection of Cyanoboletus (ASP-82, B-28, MF373585) among selected members of related boletoid genera.
The tree with the highest log likelihood (-4486.9848) is shown. Initial tree(s) for the heuristic search were obtained automatically by applying Neighbor-Join and BioNJ algorithms to a matrix of pairwise distances estimated using the Maximum Composite Likelihood (MCL) approach, and then selecting the topology with superior log likelihood value. A discrete Gamma distribution was used to model evolutionary rate differences among sites [5 categories (+G, parameter = 0.2633)]. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 26 nucleotide sequences. There were a total of 1027 positions in the final dataset. Bootstrap support values >50 are shown along the branches.
7 Publications
4.2 Publication 2: A unique arsenic speciation profile in Elaphomyces spp. ("deer truffles") - trimethylarsine oxide and methylarsonous acid as significant arsenic compounds.
63 Analytical and Bioanalytical Chemistry (2018) 410:2283 –2290 https://doi.org/10.1007/s00216-018-0903-3
COMMUNICATION
A unique arsenic speciation profile in Elaphomyces spp. ( Bdeer truffles ^) —trimethylarsine oxide and methylarsonous acid as significant arsenic compounds
Simone Braeuer 1 & Jan Borovi čka 2,3 & Walter Goessler 1
Received: 31 October 2017 /Revised: 21 December 2017 /Accepted: 18 January 2018 /Published online: 12 February 2018 # The Author(s) 2018. This article is an open access publication
Abstract Arsenic and its species were investigated for the first time in nine collections of Elaphomyces spp. ( Bdeer truffles ^) from the Czech Republic with inductively coupled plasma mass spectrometry (ICPMS) and high-performance liquid chromatography coupled to ICPMS. Th e total arsenic concentrations ranged from 12 to 42 mg kg −1 dry mass in samples of E. asperulus and from 120 to 660 mg kg −1 dry mass in E. granulatus and E. muricatus . These concentrations are remarkably high for terrestrial organisms and demonstrate the arsenic-accumulating ability of these fungi. The dominating arsenic species in all samples was methylarsonic acid which accounted for more than 30% of the extractable arsenic. Arsenobetaine, dimethylarsinic acid, and inorganic arsenic were present as well, but only at trace concentrations. Surprisingly, we found high amounts of trimethylarsine oxide in a ll samples (0.32 –28% of the extractable arsenic). Even more remarkable was that all but two samples contained significant amounts of the highly toxic trivalent arsenic compound methylarsonous acid (0.08 –0.73% of the extractable arsenic). This is the first report of the occurrence of trimethylarsine oxide and methylarsonous acid at significant concentrations in a terrestrial organism. Our findings point out that there is still a lot to be understood about the biotransformation pathways of arsenic in the terrestrial environment.
Keywords Elaphomyces . Fungi . Deer truffles . Arsenic speciation . Trimethylarsine oxide . Methylarsonous acid
Introduction element ’s species. In water, the main arsenic compounds are the two inorganic species arsenous acid [As (III)] and arsenic Arsenic is occurring in the environment in many chemical acid [As(V)] [ 1], which possess high acute and also chronic forms. Distinguishing between these different compounds is toxicity [ 2]. As (III) and As(V) are also the main arsenic spe- essential, because arsenic ’s toxicity strongly depends on the cies in soil extracts, using water (with or without small amounts of salts, for example phosphate) and/or methanol as Parts of this work were presented at Anakon 2017 in Tübingen, Germany. extracting solution [ 3]. Additionally, small amounts of organic Electronic supplementary material The online version of this article arsenic compounds such as methylarsonic acid (MA), (https://doi.org/10.1007/s00216-018-0903-3 ) contains supplementary dimethylarsinic acid (DMA), arsenobetaine (AB), or material, which is available to authorized users. trimethylarsine oxide (TMAO) can sometimes also be found in soils [ 4]. The extractable arsenic from plants is in most * Walter Goessler cases predominantly inorganic arsenic [ 5], although there are [email protected] also reports on MA and DMA as major arsenical in plant extracts [ 6, 7]. Significant amounts of other arsenicals like 1 Institute of Chemistry, Analytical Chemistry for Health and Environment, University of Graz, Universitaetsplatz 1, TMAO are found in plants only very rarely [ 8]. The main 8010 Graz, Austria arsenic metabolites of terrestrial mammals are MA and 2 The Czech Academy of Sciences, Nuclear Physics Institute, Hlavní DMA [ 9, 10 ]. In rare occasions, small amounts of the trivalent 130, 25068 Husinec- Řež, Czech Republic methylated arsenic species methylarsonous acid [MA(III)] 3 The Czech Academy of Sciences, Institute of Geology, Rozvojová and dimethylarsinous acid have also been detected in urine 269, 16500 Prague 6, Czech Republic of terrestrial mammals [ 11 ]. Apart from urine, MA(III) has 2284 Braeuer S. et al. only been detected in a sample of soil that was treated with coupled plasma mass spectrometry (ICPMS) as well as their MA(V) [ 12 ] and in carrots with unusually high total arsenic arsenic speciation with high-performance liquid chromatogra- concentrations [ 13 ]. Until now, MA(III) has not been found in phy (HPLC) coupled to ICPMS. any other environmental sample. The detection of this com- pound in feed or foodstuff would be alarming, because studies have shown that MA(III) is even more toxic than As(III) [ 14 ]. Experimental In the marine ecosystem, the arsenic concentrations and arsenic speciation are completely different from the terrestrial Sample collection, identification, and preparation environment. First, the total arsenic concentrations in marine organisms are usually much higher than in terrestrial ones, and Elaphomyces samples were collected in Bohemia, second, the major arsenic species in fish or algae are AB or Czech Republic, at spruce plantations mostly from places more complex molecules like arsenosugars, or, more recently dug by wild boars; determination of species is based on mor- discovered, lipid-soluble arsenic species, which are seldom phological characters. Ascocarps were thoroughly brushed in found in significant concentrations in terrestrial samples [ 15 , distilled water and frozen. Six samples (sample IDs: ASP-44, 16 ]. One big exception is macrofungi. Depending on the fun- ASP-55, ASP-57, ASP-56, ASP-58, and ASP-59) were lyoph- gal species, they can have similarly high concentrations of ilized. One sample of E. granulatus (ID: ASP-84) and two total arsenic as marine organisms (up to more than 1000 mg samples of E. asperulus (IDs: ASP-85a and ASP-85b) were kg −1 dm) [ 17 , 18 ]. Further, macrofungi are one of the few kept frozen until analysis. They were thawed, homogenized terrestrial organisms that can contain AB as main arsenic spe- with an ultra-centrifugal mill (ZM200, 1 mm titanium sieve, cies [ 19 , 20 ]. In other fungal species, inorganic arsenic and Retsch GmbH, Haan, Germany), digested, and also extracted DMA are the dominating arsenic compounds [ 21 , 22 ]. MA is within 1 day. The water content was determined in a drying most often only present at very low concentrations, or even oven at 100 °C for around 16 h. not at all, and only rarely a major constituent of the fungal arsenic speciation [ 19 ]. Up to now, TMAO, arsenocholine Determination of total element concentrations (AC), the tetramethyl arsonium ion (TETRA), and arsenosugars have been found much more seldom in All homogenized samples were digested in a microwave heat- macrofungi, and mostly only at trace concentrations [ 22 ]. ed pressurized digestion system (Ultraclave 4, MLS GmbH, There are thousands of macrofungal species in temperate Leutkirch, Germany). Each sample was prepared in triplicates. ecosystems, but the arsenic speciation has been investigated First, about 100 mg (weighed to 0.1 mg) of the samples was only in a very small part of it so far. For example, there is no put into quartz vessels. 5 mL nitric acid ( ≥ 65% m/m p.a., Carl information at all about arsenic compounds in hypogeous fun- Roth GmbH + Co.KG, Karlsruhe, Germany, further purified gi (which produce macroscopic fruit-bodies partially or via sub-boiling) was added and the vessels were closed loose- completely embedded in soil or humus, truffle-like fungi), ly with PTFE-caps. The digestion oven was loaded with and even data on the total arsenic concentrations in these or- 4.0*10 6 Pa of argon (5.0, Messer, Gumpoldskirchen, ganisms are scarce. To the best of our knowledge, there are Austria) and then heated up to 250 °C. The temperature was only two publications on arsenic in hypogeous fungi; one is held for 30 min, and then the system was cooled down a gain. written by Orczán et al., who investigated 22 elements, includ- The digests were transferred to 50 mL PP-tubes (Greiner Bio- ing arsenic, in 17 different hypogeous fungi species, 93 sam- one, Kremsmünster, Austria) and diluted with ultrapure water ples in total [ 23 ]. They found on average 4 ± 12 mg As kg −1 (18.2 M Ω*cm, Merck Millipore, Bedford, USA) to a final dry mass (dm) in these samples (3 ± 10 mg As kg −1 dm when volume of 50 mL (final concentration of nitric acid: 10% looking at Elaphomyces spp . only, n = 50). The second report v/v). For quality control, the Standard Reference Materials® is by Ljubojevic et al., who found 4.4 mg As kg −1 in soil and (SRM) 1573a (Tomato Leaves, NIST, Gaithersburg, USA, 2.1 mg As kg −1 (probably fresh mass) in Choiromyces n = 15) and SRM® 1568b (Rice Flour, n = 5) were digested meandriformis [24 ]. together with the samples as well as blanks ( n = 22). Elaphomyces (Bdeer truffles ^) is one of the most important The element concentrations were determined with an in- ectomycorrhizal fungal genera in temperate and subarctic for- ductively coupled plasma triple quadrupole mass spectrometer est ecosystems [ 25 ]. Preliminary neutron activation screening (ICPQQQMS, Agilent 8800, Agilent Technologies, of arsenic in ascocarps (fruit-bodies) of several Elaphomyces Waldbronn, Germany). The instrument was equipped with a species revealed elevated arsenic concentrations, up to hun- MicroMist nebulizer, a Scott-type spray chamber, and Cu/Ni dreds of mg kg −1 dm. For this reason, we collected several cones. The following elements were analyzed in the samples: ascocarps of three Elaphomyces species ( E. granulatus, E. Ag, Al, As, B, Ba, Bi, Ca, Cd, Co, Cr, Cs, Cu, Fe, Gd, Hg, K, muricatus , and E. asperulus ) and determined the concentra- Li, Mg, Mn, Mo, Na, Ni, P, Pb, Rb, S, Sb, Se, Sn, Sr, Te, Tl, U, tions of arsenic and around 30 other elements with inductively V, Zn. The selected collision/reaction cell modes (helium, A unique arsenic speciation profile in Elaphomyces spp. ( “deer truffles ”)—trimethylarsine oxide and... 2285 hydrogen, oxygen and no cell gas) and mass to charge ratios column (150 * 4.6 mm, 5 μm, Agilent) and an aqueous pyr- and the settings of the instrument can be found in the idine solution (10 mM, pH 2.3, adjusted with nitric acid, Electronic Supplementary Material (ESM, Tables S1 and 1.5 mL min −1, 30 °C) were employed for cation-exchange S2 ). Quantification was obtained via external calibration. chromatography. The injection volume was 20 μL in both The calibration solutions were prepared in 15 mL PP-tubes methods. Ammonium dihydrogen phosphate (Suprapur (Greiner Bio-one) and consisted of 10% v/v of nitric acid ( ≥ 99.99%), ammonia solution (Suprapur 25% m/m), and pyri- 65% m/m p.a., Carl Roth GmbH + Co.KG) and aliquots of the dine (30% m/m p.a.) were obtained from Merck KGaA single element standards (Carl Roth GmbH + Co.KG). Each (Darmstadt, Germany). calibration standard contained all elements except mercury, The arsenic signal was detected with ICPQQQMS in oxy- which was prepared in separate solutions. These contained gen reaction mode at m/z 91 ( 75 As + ➔ 75 As 16 O+). 15% v/v
8% v/v nitric acid and 2% v/v hydrochloric acid (Rotipuran® CO 2 (1% v/v in Ar) was added as optional gas between spray 37% m/m, p.a., subboiled twice in-house, Carl Roth GmbH + chamber and torch to enhance the arsenic signal and compen- Co.KG) for a better stabilization of the element. sate for any possible carbon enhancement effect from the or- For quality assurance and quality control, the digested ganic matrix of the extracts. SRMs® 1573a and 1568b as well as SRM® 1640a (Trace Quantification and identification of the arsenic species Elements in Natural Water, diluted 1 + 9 with ultrapure water were achieved via external calibration (0.05 –100 μg As L −1 and 10% v/v nitric acid, n = 7) were measured with the sam- for each compound). The calibration solutions were prepared ples. After every 10th sample, a calibration standard was re- in 15 mL PP-tubes (Greiner Bio-one) with ultrapure water and measured to determine a possible drift of the instrument. aliquots of standard solutions of the different arsenic species. 200 μg L −1 of Be, Ge, In, and Lu (1% v/v nitric acid) were These standard solutions (1000 mg As L −1 each) were pre- added online via a t-piece in front of the nebulizer to all sam- pared as follows. As [V] was prepared from Na 2HAsO 4*7 ples and served as internal standards. H2O, purchased from Merck (Darmstadt, Germany). Methylarsonic acid (MA) was synthesized from NaAsO 2 (pur- Arsenic speciation analysis chased from Merck) and MeI (Meyer reaction). DMA was prepared from sodium dimethylarsinate (Fluka, Buchs, For extraction, 200 mg of the samples was weighed to 0.1 mg Switzerland). MA(III) was prepared by dissolving methyl ar- into 15 mL PP-tubes (Greiner Bio-one). Each sample was senic diiodide in water with 5% v/v methanol. AB, TMAO, prepared in triplicates. Four mL of ultrapure water was added. AC and TETRA were synthesized according to literature The mixtures were shaken, put into an ultrasonic bath for [27 –30 ]. 15 min (Transsonic T 700/H, Elma GmbH&Co.KG, Singen, Due to the instability of MA(III) was not added to the Germany) and then centrifuged at 3300× g for 10 min (Rotina calibration standards, but was quantified via the calibration 420 R, Hettich Lab Technology, Tuttlingen, Germany). The of DMA. Its identity was checked by spiking the extract in extracts were filtered with syringes (Norm-Ject, Henke-Sass the following manner: 20 μL of the extract and 2 μL of a Wolf GmbH, Tuttlingen, Germany) through 0.2 μm polyam- solution of MA(III) with a 10 times higher concentration than ide syringe filters (Chromafil® Xtra PA-20/13, Macherey- in the extract were taken up by the injector needle and then Nagel GmbH & Co. KG, Düren, Germany). One part of one injected together onto the column. Additionally, the disappear- filtered replicate of each sample was mixed with 10% v/v of ance of the peak after the addition of hydrogen peroxide con- hydrogen peroxide (Rotipuran®, 30% m/m p.a., stabilized, firmed the initial presence of MA(III). Carl Roth GmbH + Co.KG), and put into an oven for 1 h at During anion- and also during cation-exchange chromatog- 45 °C. raphy, one calibration standard was re-measured after every Arsenic speciation analysis was carried out with HPLC- 10th sample for stability control. Since there is no certified ICPQQQMS, on the same day of extraction. The HPLC sys- reference material for arsenic species in a matrix that is com- tem consisted of an Agilent 1200 HPLC, equipped with a parable to mushrooms, we injected SRM® 1640a (Trace ele- degasser, a quaternary pump, a thermostatted autosampler, ments in natural water, n = 3) and compared the inorganic and a thermostatted column compartment. We applied anion- arsenic concentration with the certified value for total arsenic. exchange chromatography for the determination of As(V), The extraction efficiency was determined by diluting all DMA, MA and MA(III), and cation-exchange chromatogra- extracts with 1% v/v nitric acid and then measuring the arsenic phy for the determination of AB, TMAO, AC, and TETRA. signal with ICPQQQMS (m/z 75 ➔91, oxygen mode, plus
The methods have been validated elsewhere [ 26 ]. For anion- 15% v/v CO 2 as optional gas). Quantification was obtained exchange chromatography, a PRP-X100 column (150 * with external calibration. 4.6 mm, 5 μm, Hamilton, Bonaduz, Switzerland) and an aque- The identity of TMAO in the extracts was verified via ous phosphate buffer (20 mM, pH 6.0, adjusted with ammo- HPLC- electrospray ionization mass spectrometry (ES-MS, nia, 1 mL min −1, 40 °C) were used. A Zorbax 300-SCX 6120, Agilent Technologies). Again, the cation-exchange 2286 Braeuer S. et al. column Zorbax 300-SCX was employed with 0.5 M formic concentrations. Surprisingly, there were significant amounts acid and 0.03 M ammonium formate (pH = 2.3) and 8% v/v of MA(III) in the extracts of E. granulatus and E. muricatus , methanol as mobile phase. The flow rate was 1.5 mL min −1, confirmed by spiking and oxidation experiments (as described and the flow was split via a T-piece after the column; one part in the experimental section). This compound accounted for was going to the ES-MS, and one to the waste. The injection 0.16 –0.74% of the detected arsenic species, which corre- volume was 1 μL. The settings of the ES-MS were 90 V sponds to up to 1.2 mg As kg −1 dm (see Fig. 1). Concerning fragmentor voltage, 1000 V capillary voltage, 350 °C gas E. asperulus , the compound was only detected in one of the temperature, 12 L min −1 drying gas. TMAO was recorded in samples (around 0.01% of the arsenic species). We also found + the SIM mode at a m/z ratio 137 ((CH 3)3AsOH ). small amounts of some unknown arsenic species (in total less than 1% of the arsenic species). One of these compounds was even eluting after around 11 min on the cation-exchange col- Results umn (see Fig. 1c), which is very late compared to the most strongly retained known arsenic species, TETRA, with The results for all reference materials were generally in good 6.5 min. Overall, there were no apparent differences between agreement with the certified values, as can be found in ESM the fresh and the dried samples. Table S3 . The water content of the three fresh samples was between 49 and 52%. In order to be able to compare the results with the Discussion dried samples, the individual water content values were used to convert the results of the fresh samples into concentrations The total arsenic concentrations in our samples were quite on a dry mass basis. high, namely up to 660 mg kg −1 dm. When compared to other The total arsenic concentrations in the samples ranged from macrofungi, our results are not on top of the arsenic accumu- 12 to 660 mg kg −1 dm. The three samples of E. asperulus lating species, but certainly in the upper part of the ranking contained between 12 and 42 mg As kg −1 dm, whereas the [17 ]. The only two other publications on arsenic in hypogeous arsenic concentration in the four samples of E. granulatus fungi that we are aware of found less than 10 mg As kg −1 [23 , ranged from 120 to 660 mg kg −1 dm. The two samples of 24 ], which is almost 100 times lower than the total arsenic E. muricatus contained 180 ± 30 and 280 ± 10 mg kg −1 dm. concentrations in our samples of E. granulatus and Interestingly, the concentrations of Na, K, Rb and, less pro- E. muricatus . Even the three samples of E. asperulus, which nounced, Cs were lower in E. asperulus than in the other contained 12 –42 mg kg −1 dm, were higher than these litera- samples. For example, E. asperulus contained only 37 – ture values. Only the two samples with the highest arsenic 310 mg Na kg −1 dm, whereas the other samples contained concentrations (400 and 660 mg kg −1 dm) originated from 2800 –4700 mg Na kg −1 dm. The concentrations of arsenic mining areas with probably elevated arsenic concentrations and the alkali metals are listed in Table 1. All other elements in soil, while all other samples came from pristine regions can be found in ESM Table S4 . and still contained up to 280 mg kg −1 dm. To find the reason The extraction efficiencies were 83 ± 6% for E. granulatus , for this discrepancy with the two other studies [ 23 , 24 ], cer- around 80 and 54% for the two samples of E. muricatus , and tainly more samples will have to be investigated. only 3 –14% for E. asperulus. The mean column recovery over Perhaps the most striking discovery of our study is the pres- all samples was 85 ± 10% (range 65 –99%). Taken together ence of significant amounts of MA(III) in most of the extracts. with the extraction efficiency, this means that we were able This compound has never been found in mushrooms before, to detect and quantify with arsenic speciation analysis only 2 – and also the reports of MA(III) in other samples are very rare 11% of the total arsenic in E. asperulus and 49 –82% in the [11 –13 ]. It has to be noted that the correct detection and quan- other samples. tification of this molecule has proven to be very tricky, because The major arsenic species in all samples was MA, which of its lability and quick oxidation to the pentavalent equivalent accounted for 80 ± 20% of the sum of all arsenic species that [31 , 32 ]. On the other hand, in the case of the detection of were detected with HPLC-ICPMS. The second most abundant MA(III) in carrots [ 13 ], there is a slight possibility that small arsenic compound was TMAO, ranging from 0.37 to 37%, amounts of the originally present pentavalent MAwere reduced with a median of 17%. In absolute concentrations, this means to MA(III) during extraction at elevated temperatures (60 °C). 0.15 –40 mg kg −1 dm. The identity of TMAO in the extracts Of course, one cannot exclude to 100% that this also applies to was confirmed by HPLC-ES-MS; the chromatogram is pro- our investigated Elaphomyces samples, but using pure water at vided in ESM Fig. S1 . Inorganic arsenic accounted for 1 – room temperature as extraction agent was specifically chosen to 3.5% of the arsenic species in E. asperulus and only for influence the original arsenic speciation as little as possible. around 0.01% of the arsenic species in the other samples. Because of the quick oxidation of MA(III), it is possible DMA and AB were generally only present at trace that the concentration of MA(III) in our investigated samples nqeasncseito rfl in profile speciation arsenic unique A
Table 1 Concentrations of total arsenic (mg kg −1 dm), extracted arsenic (mg kg −1 dm and % of the total arsenic in brackets), sum of all arsenic species (mg kg −1 dm and % of the extracted arsenic in brackets), arsenic species (mg kg −1 dm), and the alkali elements (mg kg −1 dm) in the investigated samples of Elaphomyces Elaphomyces Sample ID ASP-44 ASP-55 ASP-57 ASP-84 ASP-56 ASP-58 ASP-59 ASP-85a ASP-85b Species E. granulatus E. granulatus E. granulatus E. granulatus E. muricatus E. muricatus E. asperulus E. asperulus E. asperulus State when analyzed Dried Dried Dried Fresh Dried Dried Dried Fresh Fresh
Total As 151 ± 8 400 ± 30 660 ± 30 120 ± 7 180 ± 30 280 ± 10 12 ± 1 18 ± 1 42 ± 1 (spp. Extracted As 130 ± 10 330 ± 3 514 ± 8 110 ± 20 145 ± 1 150 ± 3 1.8 ± 0.2 1.1 ± 0.1 1.3 ± 0.09 “ (83 ± 9%) (81.6 ± 0.7%) (77 ± 1%) (90 ± 10%) (79.6 ± 0.8%) (54 ± 1%) (14 ± 2%) (6.3 ± 0.8%) (3.1 ± 0.2%) trufflesdeer
Sum of species 130 ± 20 280 ± 40 440 ± 40 94 ± 5 (90 ± 20%) 132 ± 5 (91 ± 4%) 136 ± 6 1.4 ± 0.3 0.9 ± 0.2 0.80 ± 0.1
(99 ± 2%) (85 ± 10%) (85 ± 9%) (91 ± 5%) (80 ± 10%) (80 ± 20%) (65 ± 10%) ” ) MA 100 ± 10 280 ± 30 420 ± 40 93 ± 6 130 ± 5 94 ± 3 0.86 ± 0.09 0.7 ± 0.2 0.48 ± 0.05 — rmtyasn xd n..2287 and... oxide trimethylarsine TMAO 22 ± 3 2.1 ± 0.3 9.9 ± 0.7 0.34 ± 0.03 0.7 ± 0.2 42 ± 3 0.5 ± 0.2 0.15 ± 0.03 0.31 ± 0.04 MA (III) 0.9 ± 0.2 1.2 ± 0.2 0.72 ± 0.09 0.9 ± 0.3 0.62 ± 0.08 0.22 ± 0.05 ~ 0.01 < 0.002 < 0.002 DMA ~ 0.05 ~ 0.03 0.2 ± 0.1 < 0.002 ~ 0.03 0.21 ± 0.02 ~ 0.02 ~ 0.01 ~ 0.01 AB 0.16 ± 0.04 ~ 0.05 ~ 0.06 < 0.002 ~ 0.06 0.2 ± 0.1 ~ 0.008 ~ 0.003 ~ 0.004 As (V) ~ 0.02 ~ 0.05 0.09 ± 0.03 ~ 0.02 ~ 0.05 ~ 0.04 ~ 0.03 ~ 0.004 ~ 0.003
Unkown species ~ 0.1 ~ 0.4 ~ 0.7 ~ 0.04 ~ 0.2 ~ 0.6 ~ 0.004 < 0.002 ~ 0.01 (sum) Li 0.019 ± 0.005 0.056 ± 0.002 0.082 ± 0.001 0.078 ± 0.005 0.021 ± 0.003 0.0454 ± 0.0009 0.01 ± 0.001 0.019 ± 0.001 0.048 ± 0.004 Na 3600 ± 200 4100 ± 400 3200 ± 100 3600 ± 200 2800 ± 200 4700 ± 300 310 ± 20 37 ± 2 60 ± 20 K 8200 ± 100 15,000 ± 2000 24,800 ± 700 16,100 ± 900 16,000 ± 1000 3900 ± 300 2500 ± 200 1380 ± 70 950 ± 60 Rb 611 ± 8 340 ± 20 820 ± 50 177 ± 9 580 ± 30 510 ± 30 108 ± 4 19 ± 1 13.9 ± 0.8 Cs 42.3 ± 0.8 73 ± 3 104 ± 1 14.6 ± 0.5 21.5 ± 0.7 51.5 ± 0.6 10.5 ± 0.2 3.5 ± 0.2 3.3 ± 0.1 2288 Braeuer S. et al.
Further, the dominating arsenic species in all extracts was MA, which has already been reported for a few other fungi, like Sarcosphaera coronaria [19 ], which is also an ascomy- cete. However, in most macrofungi, MA is only a minor arse- nic compound or even not present at all [ 22 ]. The second most abundant arsenic compound in our samples was TMAO, ac- counting for up to 37% of the sum of arsenic species that were detected with HPLC-ICPMS or, in other words, up to 15% of the total arsenic. Already in 1945, Challenger proposed a transformation pathway of inorganic arsenic by the filamentous fungus Scopulariopsis brevicaulis with consecutive reduction and methylation steps, via the penta- and trivalent forms of MA and DMA to TMAO and further to trimethylarsine (TMA) [33 ]. For humans and other terrestrial mammals, this is not directly applicable, because TMAO is hardly ever found in mammals ’ urine. Hence, alternate mechanisms have been pro- posed, with DMA as final product [ 34 ]. Many macrofungi also contain DMA as main arsenic species, but in our investi- gated extracts of Elaphomyces spp., this compound is only present at trace concentrations. Instead, MA and TMAO make up for more than 90% of the speciated arsenic. One can spec- ulate that the transformation of arsenic in these fungi could actually be quite close to the pathway described by Challenger. DMAwould only be an intermediate that is quick- ly further methylated to TMAO and/or TMA. The latter one is actually volatile and has a very distinct smell. One could spec- ulate that the hypogeous fungi are actively producing TMA (via TMAO) to attract wild boars and other mycophagous mammals. On the other hand, the ingestion of Elaphomyces spp . might pose an increased health risk for the animals, be- cause MA(III) is highly toxic [ 14 ]. It has to be noted that there is no clear evidence that arsenic is transformed by macrofungi. Alternatively, associated mi- crobiota could be responsible for the formation of the different arsenic compounds, which may be subsequently taken up by the fungi, but this theory is not proven either. Interestingly, the extraction efficiencies were acceptable, though not 100%, for E. granulatus and E. muricatus , but less than 15% for E. asperulus . This means that there is still a large part of the fungal arsenic of which we do not know the chem- ical form. Since this arsenic is not extractable with water, one possibility could be lipid-soluble arsenicals. Another option Fig. 1 a Anion-exchange chromatograms of an extract (solid line) and of a standard, containing 5 μg As L −1 of AB, DMA, MA, and As(V) (dotted would be that the arsenic is strongly attached or bound to large line). b Anion-exchange chromatograms of an extract; pure (solid line) bio-molecules, such as proteins. Additional extraction exper- and spiked with MA(III) (dotted line). c Cation-exchange chromatograms iments will be necessary to elucidate this question in the −1 of an extract (solid line) and of a standard, containing 5 μg As L of AB, future. TMAO, AC, and TETRA (dotted line) is even underestimated. Regardless of the actual original con- Conclusion centrations of MA(III) in the samples, its pure presence is a unique discovery in the field of arsenic speciation in the The investigated species of Elaphomyces are not only accu- environment. mulating arsenic, but also possess a unique arsenic speciation; A unique arsenic speciation profile in Elaphomyces spp. ( “deer truffles ”)—trimethylarsine oxide and... 2289 the two major arsenic compounds in the extracts were MA and 7. Ruiz-Chancho MJ, López-Sánchez JF, Schmeisser E, Goessler W, TMAO and also significant amounts of MA(III) were detect- Francesconi KA, Rubio R. Arsenic speciation in plants growing in arsenic-contaminated sites. Chemosphere. 2008; https://doi.org/10. ed. This is indicating that the arsenic metabolism of these 1016/j.chemosphere.2007.11.054 . organisms is very different from all other organisms that have 8. Ruiz-Chancho MJ, López-Sánchez JF, Rubio R. Occurrence of been investigated so far. The reason for this is not clear at all. methylated arsenic species in parts of plants growing in polluted One very speculative hypothesis is that TMAO is further me- soils. Int J Environ Anal Chem. 2011; https://doi.org/10.1080/ 03067310903243944 . tabolized to TMA, which is then used for attracting mycoph- 9. Vahter M. Mechanisms of arsenic biotransformation. Toxicology. agous mammals. On the other hand, the presence of MA(III) 2002; https://doi.org/10.1016/S0300-483X(02)00285-8 . might be a health risk for wild animals that feed on these 10. Watanabe T, Hirano S. Metabolism of arsenic and its toxicological mushrooms. Overall, our investigations show that definitely relevance. Arch Toxicol. 2013; https://doi.org/10.1007/s00204- 012-0904-5 . more work is needed to elucidate the role of arsenic in the 11. Aposhian HV, Gurzau ES, Le XC, Gurzau A, Healy SM, Lu X, terrestrial environment and its interactions with macrofungi. et al. Occurrence of monomethylarsonous acid in urine of humans exposed to inorganic arsenic. Chem Res Toxicol. 2000; https://doi. Acknowledgements Open access funding provided by Austrian Science org/10.1021/tx000114o . Fund (FWF). This research was supported by the joint project FWF I 12. Száková J, Tlusto š P, Goessler W, Pavlíková D, Schmeisser E. 2352-B21 (Austrian Science Fund) – GA ČR GF16-34839L (Czech Response of pepper plants (Capsicum annum L.) on soil amend- Science Foundation). Institutional support (Jan Borovi čka) was received ment by inorganic and organic compounds of arsenic. Arch Environ from the projects RVO61389005 and RVO67985831. 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Electronic Supplementary Material
A unique arsenic speciation profile in Elaphomyces spp. (“deer truffles”)
– trimethylarsine oxide and methylarsonous acid as significant arsenic compounds
Simone Braeuer, Jan Borovička, Walter Goessler
Additional files available under 10.1007/s00216-018-0903-3 . Table S1 All measured elements with the elected octopole gas modes and m/z ratios
octopole Element Name m/z gas mode Li nogas 7 B nogas 11 Na He 23 Mg He 24 Al nogas 27 P O2 31 -> 47 S O2 32 -> 48 K He 39 Ca He 43 V He 51 Cr He 52 Mn He 55 Fe He 56 Co He 59 Ni He 60 Cu He 65 Zn He 66 As O2 75 -> 91 Se H2 78 Rb He 85 Sr He 88 Mo nogas 98 Ag nogas 107 Cd nogas 111 Sn nogas 118 Sb nogas 121 Te nogas 125 Cs he 133 Ba nogas 137 Gd nogas 157 Hg nogas 201 Tl nogas 205 Pb nogas 208 Bi nogas 209 U nogas 238 Table S2 Configuration of ICPQQQMS. Differing settings during HPLC- ICPMS in O 2-mode in brackets O2 H2 He nogas Scan Type MS/MS Single Quad Single Quad Single Quad RF Power [W] 1600 1600 1600 1600 RF Matching [V] 1.8 1 .8 1 .8 1 .8 Smpl Depth [mm] 8 8 8 8 Carrier Gas [L/min] 1.1 (0.85) 1.1 1 .1 1 .1 Option Gas [%] 0 (15) 0 0 0 Nebulizer Pump [rps] 0.1 (0.5) 0.1 0 .1 0 .1 S/C Temp [°C] 2 2 2 2 Extract 1 [V] 0 0 -1 .5 -1 .5 Extract 2 [V] -160 -150 -195 -185 Omega Bias [V] -90 -80 -110 -95 Omega Lens [V] 6.6 6 .6 7 .9 7 .9 Q1 Entrance [V] -1 -1 -3 0 Q1 Exit [V] -1 -1 -1 -1 Cell Focus [V] 2 -4 -3 -6 Cell Entrance [V] -50 -50 -50 -50 Cell Exit [V] -60 -60 -60 -60 Deflect [V] 3.2 -60 -6 13 Plate Bias [V] -60 -60 -60 -60 Q1 Bias [V] -2 -4 -4 -3 Q1 Prefilter Bias [V] -44 -36 -48 -40 Q1 Postfilter Bias [V] -18 -18 -34 -2 He flow [mL/min] 0 0 4 0 H2 flow [mL/min] 0 5.5 0 0 4th cell gas flow [%] 25 0 0 0 OcP Bias [V] -5 -18 -18 -8 OcP RF [V] 190 200 130 120 Energy discrimination [V] -7 0 3 5
Table S3 and Table S4 available under „Supplementary m aterial “ + Fig. S1 HPLC-ES-MS chromatograms of m/z 137 ((CH 3)3AsOH ). Solid line = extract of ASP-058, diluted 1+9 with water. Dotted line = same sample as for the solid line, spiked with a pure solution of TMAO (200 µg As L -1 ). Dashed line = same sample and chromatographic method as for the solid line, but with ICPMS (ICPQQQMS 8800, oxygen mode, m/z 7 5 91) as detector instead of ES-MS Table S3 Results for the reference materials (n.c. = not certified) SRM 1640a certified [µg L-1] measured [µg L-1] Li 0.40034 ± 0.0092 (reference value only) 0.38 ± 0.06 B 300.7 ± 3.1 280 ± 20 Na 3112 ± 31 (reference value only) 2900 ± 300 Mg 1050.2 ± 3.4 (reference value only) 990 ± 60 Al 52.6 ± 1.8 53 ± 7 P n.c. n.c. S n.c. n.c. K 575.3 ± 2 (reference value only) 583 ± 8 Ca 5570 ± 16 (reference value only) 6000 ± 300 V 14.93 ± 0.21 15.1 ± 0.9 Cr 40.22 ± 0.28 39 ± 2 Mn 40.07 ± 0.35 41 ± 2 Fe 36.5 ± 1.7 40 ± 3 Co 20.08 ± 0.24 19 ± 2 Ni 25.12 ± 0.12 24 ± 1 Cu 85.07 ± 0.48 82 ± 6 Zn 55.2 ± 0.32 53 ± 4 As 8.01 ± 0.067 7.7 ± 0.5 (speciation analysis: 8.0 ± 0.2) Se 19.97 ± 0.16 19.1 ± 0.6 Rb 1.188 ± 0.011 (reference value only) 1.3 ± 0.1 Sr 125.03 ± 0.86 118 ± 7 Mo 45.24 ± 0.59 42 ± 4 Ag 8.017 ± 0.042 7 ± 1 Cd 3.961 ± 0.072 3.8 ± 0.3 Sn n.c. n.c. Sb 5.064 ± 0.045 4.87 ± 0.1 Cs n.c. n.c. Ba 150.6 ± 0.74 139 ± 4 Gd n.c. n.c. Hg n.c. n.c. Tl 1.606 ± 0.015 1.5 ± 0.2 Pb 12.005 ± 0.04 11.2 ± 0.7 U 25.15 ± 0.26 23 ± 1 SRM 1573a certified [µg kg-1] measured [µg kg-1] Li n.c. n.c. B 33300 ± 700 29000 ± 1000 Na 136000 ± 4000 124000 ± 6000 Mg 12000000 (noncertified) 10600000 ± 700000 Al 598000 ± 12000 470000 ± 20000 P 2160000 ± 40000 2400000 ± 300000 S 9600000 (noncertified) 10000000 ± 1000000 K 27000000 ± 500000 29000000 ± 2000000 Ca 50500000 ± 900000 52000000 ± 4000000 V 835 ± 10 730 ± 50 Cr 1990 ± 60 1850 ± 80 Mn 246000 ± 8000 250000 ± 30000 Fe 368000 ± 7000 350000 ± 20000 Co 570 ± 20 490 ± 20 Ni 1590 ± 70 1420 ± 40 Cu 4700 ± 140 4000 ± 100 Zn 30900 ± 700 31000 ± 2000 As 112 ± 4 120 ± 10 Se 54 ± 3 72 ± 7 (interference of doubly charged Sm) Rb 14890 ± 270 15000 ± 1000 Sr 85000 (noncertified) 90000 ± 6000 Mo 460 (noncertified) 400 ± 30 Ag 17 (noncertified) 15 ± 2 Cd 1520 ± 40 1490 ± 60 Sn n.c. n.c. Sb 63 ± 6 46 ± 3 Cs 53 (noncertified) 48 ± 4 Ba 63000 (noncertified) 57000 ± 3000 Gd 170 (noncertified) 160 ± 80 Hg 34 ± 4 31 ± 4 Tl n.c. n.c. Pb n.c. n.c. U 35 (noncertified) 30 ± 6 SRM 1568b certified [µg kg-1] measured [µg kg-1] Li n.c. n.c. B n.c. n.c. Na 6740 ± 190 8400 ± 600 Mg 559000 ± 10000 580000 ± 20000 Al 4210 ± 340 3900 ± 300 P 1530000 ± 40000 1360000 ± 20000 S 1200000 ± 10000 920000 ± 20000 K 1282000 ± 11000 1230000 ± 20000 Ca 118400 ± 3100 116000 ± 6000 V n.c. n.c. Cr n.c. n.c. Mn 19200 ± 1800 19100 ± 100 Fe 7420 ± 440 7400 ± 300 Co 17.7 ± 0.5 (reference value only) 17 ± 1 Ni n.c. n.c. Cu 2350 ± 160 2300 ± 100 Zn 19420 ± 260 19100 ± 700 As 285 ± 14 295 ± 10 Se 365 ± 29 360 ± 30 Rb 6198 ± 26 6300 ± 500 Sr n.c. n.c. Mo 1451 ± 48 1400 ± 200 Ag n.c. n.c. Cd 22.4 ± 1.3 24 ± 1 Sn 5 ± 1 (reference value only) 4.6 ± 0.7 Sb n.c. n.c. Cs n.c. n.c. Ba n.c. n.c. Gd n.c. n.c. Hg 5.91 ± 0.36 10 ± 10 Tl n.c. n.c. Pb 8 ± 3 (reference value only) 7.8 ± 0.2 U n.c. n.c. Table S4 Concentrations of all measured elements in mg kg -1 dm for each investigated sample 3 ± 2 3 ± Fresh 42 ± 1 42 ± 2 77 ± < 0.01 < 60 ± 20 60 ± 8 140 ± 2 122 ± 10 70 ± < 0.001 < 0.001 < 0.001 < ASP-85b 310 ± 20 310 ± 60 950 ± 0.1 0.9 ± 0.2 2.4 ± 10 250 ± 10 210 ± 0.1 0.4 ± 0.1 3.3 ± 0.1 1.7 ± 13.9 ± 0.8 13.9 ± 0.05 1.1 ± 14.6 ± 0.8 14.6 ± 2400 ± 100 2400 ± 200 3600 ± 0.02 0.72 ± 0.65 ± 0.03 0.65 ± 0.05 ± 0.003 0.05 ± E. asperulus 0.048 ± 0.004 0.048 ± 0.813 ± 0.008 0.813 ± 0.001 0.025 ± 0.003 0.024 ± 0.0016 ± 0.0004 0.0016 ± 0.0009 0.0061 ± 0.0006 0.0169 ± Fresh 37 ± 2 37 ± 1 12 ± 6 45 ± 1 18 ± 1 19 ± 3 47 ± < 0.01 < 90 ± 20 90 ± 7 167 ± < 0.001 < 0.001 < 0.001 < ASP-85a 130 ± 10 130 ± 20 320 ± 0.9 1.3 ± 0.4 1.6 ± 10 190 ± 0.2 3.5 ± 1380 ± 70 1380 ± 0.2 ± 0.02 0.2 ± 0.53 ± 0.01 0.53 ± 100 2600 ± 200 3200 ± 0.05 0.55 ± 0.05 0.78 ± 0.03 0.47 ± 0.03 0.44 ± 0.03 0.03 ± 0.04 0.86 ± E. asperulus 0.019 ± 0.001 0.019 ± 0.043 ± 0.003 0.043 ± 0.0126 ± 0.0005 0.0126 ± 0.0191 ± 0.0008 0.0191 ± 0.0004 0.0026 ± 0.0007 0.0032 ± Dried 41 ± 3 41 ± 6 35 ± 1 12 ± ASP-59 108 ± 4 108 ± 180 ± 9 180 ± 7 201 ± < 0.001 < 310 ± 20 310 ± 0.1 4.9 ± 0.2 2.1 ± 28.9 ± 0.4 28.9 ± 0.3 11.1 ± 0.2 10.5 ± 1.04 ± 0.07 1.04 ± 200 2500 ± 0.9 643.1 ± 300 4400 ± 200 2500 ± 0.03 0.19 ± 0.05 0.22 ± 0.03 0.94 ± 0.05 0.47 ± 0.01 0.32 ± 0.01 ± 0.001 0.01 ± 0.01 0.063 ± 0.001 0.01 ± 0.006 0.15 ± E. asperulus 0.053 ± 0.003 0.053 ± 0.001 0.015 ± 0.002 0.039 ± 0.0067 ± 0.0007 0.0067 ± 0.0001 0.0058 ± 0.0002 0.0041 ± 0.0002 0.0039 ± 0.0013 ± 0.00005 0.0013 ± Dried 43 ± 3 43 ± 2 40 ± 1 34 ± ASP-58 117 ± 4 117 ± 7 179 ± 13 ± 0.6 13 ± 2.8 ± 0.1 2.8 ± 10 280 ± 30 510 ± 20.1 ± 0.9 20.1 ± 80 1450 ± 0.6 51.5 ± 4700 ± 300 4700 ± 200 1200 ± 500 5000 ± 300 3900 ± 0.07 0.88 ± 0.01 0.23 ± 0.02 0.36 ± 0.04 0.62 ± 0.63 ± 0.03 0.63 ± 0.717 ± 0.01 0.717 ± 0.01 0.008 ± E. muricatus 0.224 ± 0.007 0.224 ± 0.005 0.035 ± 0.003 0.114 ± 0.001 0.093 ± 0.619 ± 0.006 0.619 ± 0.136 ± 0.0007 0.136 ± 0.0003 0.009 ± 0.0454 ± 0.0009 0.0454 ± 0.0009 0.0145 ± 0.0005 0.0146 ± 0.0003 0.0055 ± 0.00235 ± 0.00009 0.00235 ± Dried 72 ± 5 72 ± 78 ± 6 78 ± 3 30 ± 1 24 ± 2 45 ± ASP-56 181 ± 8 181 ± < 0.001 < 180 ± 30 180 ± 30 580 ± 2.8 ± 0.04 2.8 ± 0.7 21.5 ± 0.03 0.4 ± 2800 ± 200 2800 ± 100 1400 ± 400 3500 ± 500 4800 ± 0.07 0.58 ± 0.06 0.19 ± 0.01 0.46 ± 0.03 1.12 ± 0.06 0.86 ± 0.01 0.16 ± 0.09 3.23 ± 0.01 0.64 ± 0.02 0.37 ± 0.07 ± 0.002 0.07 ± E. muricatus 0.021 ± 0.003 0.021 ± 1000 16000 ± 0.006 0.039 ± 0.001 0.079 ± 0.003 0.014 ± 0.0165 ± 0.0007 0.0165 ± 0.0002 0.0058 ± 0.0003 0.0038 ± 0.0001 0.0065 ± Fresh 26 ± 2 26 ± < 0.01 < ASP-84 114 ± 7 114 ± 7 104 ± 9 159 ± 7 120 ± 9 177 ± < 0.001 < 0.001 < 0.001 < 0.001 < 6.7 ± 0.4 6.7 ± 30 350 ± 0.1 1.2 ± 10 130 ± 80 890 ± 0.4 4.2 ± 0.1 1.7 ± 0.3 3.7 ± 0.4 3.4 ± 14.6 ± 0.5 14.6 ± 3600 ± 200 3600 ± 200 2800 ± 200 4100 ± 300 4500 ± 0.03 0.47 ± 0.05 0.97 ± 0.01 0.29 ± 16100 ± 900 16100 ± 0.046 ± 0.003 0.046 ± 0.078 ± 0.005 0.078 ± 0.004 0.142 ± 0.004 0.074 ± 0.001 0.008 ± E. granulatus 0.0068 ± 0.0006 0.0068 ± Dried 89 ± 3 89 ± 1 36 ± 2 39 ± 4 ± 0.1 4 ± ASP-57 130 ± 8 130 ± 142 ± 6 142 ± 3 170 ± 1 104 ± < 0.001 < 1 ± 0.06 1 ± 19.5 ± 1 19.5 ± 2.4 ± 0.4 2.4 ± 30 660 ± 50 820 ± 0.1 2.7 ± 1570 ± 40 1570 ± 3200 ± 100 3200 ± 200 3100 ± 200 5000 ± 0.08 1.48 ± 0.03 0.55 ± 0.02 0.42 ± 0.02 0.49 ± 0.03 1.38 ± 24800 ± 700 24800 ± 0.082 ± 0.001 0.082 ± 0.228 ± 0.004 0.228 ± 0.001 0.067 ± 0.005 0.085 ± 0.007 0.287 ± 0.005 1.163 ± E. granulatus 0.0239 ± 0.001 0.0239 ± 0.0313 ± 0.0005 0.0313 ± 0.0002 0.0142 ± 0.00299 ± 0.00006 0.00299 ± Dried 86 ± 4 86 ± 9 96 ± 3 73 ± ASP-55 72 ± 10 72 ± 2 ± 0.02 2 ± 4.5 ± 0.8 4.5 ± 20 210 ± 0.2 0.4 ± 0.3 1.2 ± 10 220 ± 30 400 ± 20 340 ± 0.2 2.1 ± 4100 ± 400 4100 ± 100 1200 ± 200 1800 ± 500 4800 ± 0.07 0.87 ± 0.05 1.99 ± 0.01 0.24 ± 0.05 0.57 ± 0.02 0.43 ± 0.02 0.08 ± 0.08 1.79 ± 0.056 ± 0.002 0.056 ± 2000 15000 ± 0.001 0.097 ± 0.002 0.037 ± 0.0008 0.01 ± 0.005 0.021 ± 0.004 0.273 ± 0.002 0.012 ± 0.409 ± 0.003 0.409 ± E. granulatus 0.0073 ± 0.0006 0.0073 ± 0.0002 0.0057 ± Dried 88 ± 2 88 ± 4 57 ± 2 75 ± ASP-44 140 ± 1 140 ± 8 151 ± 8 611 ± 136 ± 4 136 ± < 0.001 < 0.001 < 0.001 < 0.9 ± 0.2 0.9 ± 23.3 ± 0.7 23.3 ± 70 1330 ± 40 1590 ± 0.8 42.3 ± 3600 ± 200 3600 ± 4500 ± 200 4500 ± 100 8200 ± 0.03 0.61 ± 0.02 0.33 ± 0.07 4.04 ± 0.01 0.52 ± 0.01 0.77 ± 0.01 0.79 ± 0.04 0.04 ± 0.01 0.02 ± 0.05 1.58 ± 0.09 0.16 ± 0.05 0.49 ± 10.06 ± 0.09 10.06 ± 0.01 0.067 ± 0.019 ± 0.005 0.019 ± 0.009 0.103 ± E. granulatus 0.0231 ± 0.001 0.0231 ± 0.0004 0.013 ± S P K B V U Li Tl Bi Al Sr Cr Ni Cs Fe Se As Sn Sb Te Ca Zn Ba Co Cu Ag Cd Pb Rb Hg Na Gd Mg Mn Mo Species Sample ID Sample State whenState analyzed Publications
4.3 Publication 3: Homoarsenocholine - a novel arsenic compound detected for the first time in nature
80 Talanta 188 (2018) 107–110
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Talanta
journal homepage: www.elsevier.com/locate/talanta
Homoarsenocholine – A novel arsenic compound detected for the first time in nature
Simone Braeuer a, Jan Borovi čka b,c, Toma Glasnov a, Gema Guedes de la Cruz a, Kenneth B. Jensen a, Walter Goessler a,⁎ a Institute of Chemistry, University of Graz, Universitaetsplatz 1, 8010 Graz, Austria b Nuclear Physics Institute, Czech Academy of Sciences, Hlavní 130, 25068 Husinec- Řež, Czech Republic c Institute of Geology, Czech Academy of Sciences, Rozvojová 269, 16500 Prague 6, Czech Republic
ARTICLE INFO ABSTRACT
Keywords: The arsenic speciation was determined in macrofungi of the Ramaria genus with HPLC coupled to inductively Arsenic speciation coupled plasma mass spectrometry. Besides arsenic species that are already known for macrofungi, like ar- Ramaria senobetaine or arsenocholine, two compounds that were only known from marine samples so far (trimethy- Fungi larsoniopropanate and dimethylarsinoylacetate) were found for the first time in a terrestrial sample. An un- Homoarsenocholine known arsenical was isolated and identi fied as homoarsenocholine. This could be a key intermediate for further (3-hydroxypropyl) trimethylarsonium ion elucidation of the biotransformation mechanisms of arsenic. ICPMS
1. Introduction arsenic species. Concerning AB, it has been speculated that it might serve as an osmolyte and help maintaining the structure of the fruit- Macrofungi are well known for their ability to accumulate enormous bodies [7] . In a recent publication it has been shown that AB can amounts of various elements, depending on the fungal species. One of protect against osmotic and temperature-induced stress, similar to its these elements is arsenic, where more than 1000 mg kg −1 dry mass nitrogen-analogue, glycine betaine [11] . (dm) can be taken up by certain species [1 –3] . In addition to this, One unusually looking group of terrestrial macrofungi are the so- macrofungi are known to be able to contain a remarkable variety of called clavarioid fungi (coral fungi) of the genus Ramaria . Some species, organoarsenicals. Terrestrial organisms usually only contain inorganic like Ramaria flava , are edible, but there are also Ramaria fungi that can arsenic (iAs), methylarsonic acid (MA) and/or dimethylarsinic acid be poisonous to animals [12] . Until now, total arsenic concentrations (DMA) [4 –6] , but in macrofungi, arsenic species typically attributed to have been investigated in very few individual samples and range from the marine environment can be found as well. The most prominent is 0.2 to 11 mg As kg −1 dm [8,13 –19] . Only one sample of Ramaria pallida arsenobetaine (AB), which is, like iAs and DMA, the major arsenic has been investigated for its arsenic speciation so far [8] . The main compound in many macrofungi [7,8] . Trimethylarsine oxide (TMAO), arsenic species was AB (81%), followed by 13% AC and small amounts arsenocholine (AC), the tetramethylarsonium ion (TETRA) and ar- of DMA, MA and iAs. In our study, we aimed to look into the arsenic senosugars have been detected in macrofungi as well, but usually only speciation of these bizarre mushrooms to broaden the current knowl- at low or trace concentrations [7] . Up to now, it is unclear why the edge and understanding of arsenic speciation in the environment. arsenic speciation in macrofungi can vary so much between di fferent species. It is also unknown if the macrofungi are metabolizing arsenic to 2. Materials and methods the di fferent compounds themselves, if it is induced by microorganisms or if they are just accumulating it from the surrounding environment. In We investigated the arsenic speciation of six collections of the genus vitro studies by Nearing et al. have shown that AB is not present during Ramaria . Three samples were collected and identi fied by J. Borovi čka the vegetative life stage (mycelium) of Agaricus spp. , but can be found (one in Slovakia in 2014, and two in Czech Republic in 2011 and 2016), in all parts of the fungi during fruit-body formation (reproductive life and three samples were collected and identi fied by W. Goessler in stage), including the mycelium [9,10] . Another important, yet un- Austria in 2017. In order to characterize the collections we performed answered question is, why terrestrial macrofungi contain these various ITS rDNAsequencing; the sequences were submitted to the GenBank
⁎ Corresponding author. E-mail address: [email protected] (W. Goessler). https://doi.org/10.1016/j.talanta.2018.05.065 Received 10 April 2018; Accepted 19 May 2018
Available online 22 May 2018
0039-9140/ © 2018 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). S. Braeuer et al. Talanta 188 (2018) 107–110 database under the accession numbers MH366531 –MH366536. For storage, the samples were freeze-dried. Sample preparation and de- termination of the total arsenic concentration as well as of the most common water-soluble arsenic species in aqueous extracts is described in detail elsewhere [20] . Brie fly, the freeze-dried fungal samples were digested with nitric acid and then investigated with inductively coupled plasma triple quadrupole mass spectrometry (ICPQQQMS, 8800, Agi- lent Technologies, Waldbronn, Germany) for the determination of total arsenic concentrations ( Appendix A, Table S1 ). The Standard Reference Materials ® 1573a (Tomato Leaves, NIST, Gaithersburg, USA) and SRM ® 1640a (Trace Elements in Natural Water, NIST) were prepared and measured together with the samples for quality control. The results were in good accordance with the certi ficates ( Appendix A, Table S2 ). For speciation analysis, dried fungal samples were extracted with ul- trapure water and then investigated with high performance liquid chromatography (HPLC, 1200, Agilent Technologies) coupled to ICP- QQQMS. Anion-exchange and cation-exchange chromatography were used to detect and quantify arsenate [As(V)], MA, DMA, AB, TMAO, AC and TETRA. A Q-Exactive Hybrid Quadrupole-Orbitrap MS (Thermo Fisher Sci., Erlangen, Germany) was used for high-resolution electro- spray ionization mass spectrometry (HR ES-MS) measurements (Appendix A, Table S3 ). It was coupled to an HPLC with a LC cation- exchange column (Zorbax 300-SCX, Agilent Technologies) and 30 mM ammonium formate, pH 2.3% and 8% methanol as mobile phase. The flow rate of 1.5 mL min −1 was split with a T-piece after the column to reduce the input to the MS (split ratio: approximately 1 + 1).
3. Results and discussion
The total arsenic concentrations in the six investigated samples ranged from 1.7 to 61 mg kg −1 dm, with a median of 18 mg kg −1 dm (Table 1 ). Extraction with water resulted in an extraction e fficiency of Fig. 1. Exemplary anion-exchange (a) and cation-exchange (b) chromatograms 90 ± 10%, and a column recovery of 93 ± 5%. of a Ramaria extract. Dotted line: extract spiked with TMAO, TMAP, AC, TETRA The main arsenic species in the extracts was unambiguously AB, and UNK A (= AC2). accounting for 84 ± 9% of the extracted arsenic. We also detected small amounts of As(V), MA, DMA, TMAO, AC, TETRA, trimethy- Thus, UNK A was isolated by injecting an aqueous fungal extract larsoniopropanate (TMAP or AB2) and dimethylarsinoylacetate multiple times onto the cation-exchange column and collecting the re- (DMAA) in all six samples. Their identity was con firmed with spiking spective fractions. experiments and co-chromatography. The most important results are The mobile phase was removed by freeze-drying, and the residue given in Table 1 . Concentrations of all detected arsenic species can be was dissolved in a small amount of ultrapure water. The presence and found in Appendix A, Table S4 . TMAP and DMAA are known com- concentration of UNK A was controlled with HPLC-ICPMS. Next, the pounds from the marine environment [21 –23] , and DMAA has been isolate was subjected to HPLC single quadrupole ES-MS to get an idea identi fied as urinary metabolite of arsenosugars [24] , but they have on the molecular mass of the compound. At the elution time of UNK A never been found in natural terrestrial samples before. (as speci fied with HPLC-ICPMS), we found a signal with m/z 179. With Further, we found several unassigned peaks in the anion- and also this information, we started the investigation of UNK A with HR ES-MS. cation-exchange chromatograms ( Fig. 1 ). With spiking experiments, we We were able to detect a molecule with an exact m/z of 179.0411 and a excluded dimethylarsinoylethanol (DMAE), dimethylarsinoylpropio- sum formula of C 6H16 OAs. Fragmentation experiments revealed char- nate (DMAP), dimethylarsinoylbutanate (DMAB) and the glycerol-, acteristic fragments of m/z 161, 121, 105 and 59, as shown in Fig. 2 . phosphate-, sulfate- and sulfonate- arseno-riboses as possible candi- The molecular mass of 179 and the corresponding fragments have dates. Oxidation experiments proved that no known thio-arsenic com- already been reported by McSheehy et al. [25] There, the authors pounds were present. subjected a solution of inorganic arsenic and acetic acid to UV irra- One of the detected unknown compounds (UNK A in Fig. 1 b) was diation, and then investigated the solutions with ES-MS. They found attracting our attention, because it was eluting from the cation-ex- several products, including a molecule with m/z 179. We agree with change column very late, even after the permanent cation TETRA.
Table 1 Total As and extracted As concentrations in Ramaria samples [mg kg −1 dm] and detected As species [% of extracted As]. Other As species that were detected in small amounts (< 5%) are: MA, DMA, As (V), TMAO, TETRA, DMAA and several unknown As species.
Sample ID Species Origin Total As [mg kg −1] Extr. As [mg kg −1] AB [%] AC [%] TMAP [%] AC2 [%]
ASP-017 R. subbotrytis Slovakia 25 ± 2 22 ± 2 92 ± 7 1.6 ± 0.3 0.16 ± 0.02 0.27 ± 0.04 ASP-023 R. subbotrytis Czech Republic 61 ± 5 66 ± 4 91 ± 9 2.7 ± 0.4 0.3 ± 0.1 0.47 ± 0.02 ASP-068 R. subbotrytis Czech Republic 44 ± 4 39 ± 1 80 ± 20 1.7 ± 0.2 0.58 ± 0.01 0.47 ± 0.04 STM-107 R. aff. largentii Austria 1.7 ± 0.1 1.4 ± 0.1 67 ± 2 4.1 ± 0.2 1.26 ± 0.1 0.82 ± 0.08 STM-108 R. cf. pallida Austria 11.7 ± 0.2 9.5 ± 0.1 89 ± 1 1.57 ± 0.02 0.46 ± 0.02 0.37 ± 0.01 STM-109 R. cf. pallida Austria 8.3 ± 0.3 6.9 ± 0.2 87 ± 2 2.4 ± 0.1 0.83 ± 0.03 0.93 ± 0.01
108 S. Braeuer et al. Talanta 188 (2018) 107–110
arsenic [29 –31] , AC is thought to be a precursor of AB. Early in- vestigations with rat liver cells and a recent study on the function of AB showed indeed that AC can be converted to AB (and also to TMAO) [11,32,33] . Interestingly, the oldest of these works found AB aldehyde as intermediate [32] . This has not been reported in any other pub- lication since then. In analogy to AC and AB, one could assume that AC2 serves as a precursor for TMAP ( Scheme 1 ), a compound that is also present in our investigated Ramaria samples. Still, when taking a closer look at the di fferent hypothesized biotransformation mechanisms for arsenic, the existence of AC2 cannot be explained easily. Alternatively, DMAP, which is present in marine organisms [23,34] , but not in our fungal samples, or TMAP (via a hypothetical aldehyde) could be re- garded as precursors for AC2 (See Scheme 1 ), but proof for this is not existing and would have to be found through appropriate experiments.
4. Conclusions Fig. 2. Mass spectrum of HR ES-MS of UNK A (AC2). This is the first report of DMAA and TMAP in the terrestrial en- them that m/z 161 represents a water loss, m/z 121 is a protonated vironment and the overall first report of the natural occurrence of AC2. fi Me 3As, and m/z 105 is Me 2As. m/z 59 is the protonated allyl alcohol Our ndings give fresh input to the attempts to understand the geo-bio- fi corresponding to the loss of Me 3As. Thus, we identi ed UNK A as the chemical pathways of arsenic compounds. Subsequent future work (3-hydroxypropyl) trimethylarsonium ion, a homologe of AC, which we should deal with the identi fication of other small arsenic compounds in called homoarsenocholine ( “AC2 ”, structure shown in Scheme 1 ). environmental samples, which could help to complete the hypothesized For veri fication, AC2 bromide was prepared according to an up- arsenic biotransformation pathways. Possible candidates are the alde- dated literature procedure ( see Appendix A for details ) [26] . Its purity hydes of AB and TMAP or a reduced form of DMAP ( Scheme 1 ). Finally, and structure was con firmed with NMR experiments ( see Appendix A ). it has to be noted that it is very likely that AC2 was found but not Further, a solution of the pure compound was subjected to cation-ex- identi fied in other macrofungi before. When comparing published data, change HPLC-ICPMS and HR ES-MS. The results were in accordance especially chromatograms, two possible candidates of the fungal with our findings for UNK A. Successful spiking of UNK A with AC2 kingdom are Amanita muscaria and Cortinarius coalescens [35,36] . It will (and the other, known, occurring arsenic species) on HPLC-ICPMS was be interesting to verify this surmise and show that AC2 is not only our final con firmation that UNK A is indeed AC2 (See Fig. 1 b). present in fungi of the genus Ramaria . This species has never been reported in a natural sample before, and the already discussed paper by McSheehy et al. is the only one that Funding mentions the finding of AC2 in a lab experiment [25] . Homocholine, which is the nitrogen-analogue of AC2, is only occasionally investigated This work was supported by the Austrian Science Fund [FWF, grant and hardly ever discussed as a naturally occurring compound [27,28] . number I 2352-B21] and the Czech Science Foundation [GA ČR, grant According to the existing proposed biotransformation pathways of number GF16-34839L].
Scheme 1. Structures of UNK A, identi fied as homoarsenocholine, AC2, and related As species. *hypothetical, not reported yet.
109 S. Braeuer et al. Talanta 188 (2018) 107–110
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110 Appendix A Supplementary material to
Homoarsenocholine - a novel arsenic compound detected for the first time in nature
Simone Braeuer a, Jan Borovička b,c , Toma Glasnov a, Gema Guedes de la Cruz a, Kenneth B. Jensen a and Walter Goessler *a a Institute of Chemistry, University of Graz, Universitaetsplatz 1, 8010 Graz, Austria b Nuclear Physics Institute, Czech Academy of Sciences, Hlavní 130, 25068 Husinec- Řež, Czech Republic c Institute of Geology, Czech Academy of Sciences, Rozvojová 269, 16500 Prague 6, Czech Republic
* Corresponding author. E-mail adress: [email protected] Table S1. Settings of ICPQQQMS
Tune mode O2 Scan Type MS/MS Nebulizer micromist (HPLC: arimist) RF Power [W] 1550 RF Matching [V] 1.8 Smpl Depth [mm] 8 Carrier Gas [L/min] 1.1 (HPLC: 0.6) Option Gas [%] 0 (HPLC: 20) Nebulizer Pump [rps] 0.1 (HPLC: 0.4) S/C Temp [°C] 2 Makeup Gas [L/min] 0 (HPLC: 0.3) Extract 1 [V] 0 Extract 2 [V] -145 Omega Bias [V] -85 Omega Lens [V] 7.2 Q1 Entrance [V] -1 Q1 Exit [V] -1 Cell Focus [V] 2 Cell Entrance [V] -50 Cell Exit [V] -60 Deflect [V] 3 Plate Bias [V] -60 Q1 Mass Gain 130 Q1 Mass Offset 126 Q1 Axis Gain 1.0008 A1 Axis Offset 0.02 Q1 Bias [V] -2 Q1 Prefilter Bias [V] -44 Q1 Postfilter Bias [V] -26 He flow [mL/min] 0 H2 flow [mL/min] 0 4th cell gas flow [%] 25 OcP Bias [V] -5 OcP RF [V] 170 Energy discrimination [V] -7 Q2 Mass Gain 121 Q2 Mass Offset 127 Q2 Axis Gain 0.9985 Q2 Axis Offset 0.01 QP Bias [V] -12 Torch H [mm] 0.1 Torch V [mm] 0.2 EM Discriminator [mV] 4 EM Analog HV [V] 1811 EM Pulse HV [V] 1306 Table S2. Data on Certified Reference Materials (Certified and measured)
As certified measured trueness Material species [µg/kg] [µg/kg] [%] NIST 1640a total As 8.010 ± 0.067 8.0 ± 0.1 99 ± 2 NIST 1573a total As 112 ± 4 130 ± 20 120 ± 20 NIST 1640a As (V) 8.010 ± 0.067 7.9 ± 0.6 98 ± 7 Table S3. Settings of HR ES-MS (Orbitrap, equipped with a HESI source)
Spray voltage [kV] 3.5 Probe aux temp [°C] 460 Sheath gas flow [instrument units] 57 Aux gas flow [instrument units] 16 Capillary temp [°C] 280 Resolution [HWHM] 70,000 AGC target 3*10^6 max. Injection time [msec] 100 Inclusion list for MS2 experiments C4H11As, C5H13AsO1, C6H15AsO1 Resolution [HWHM] 17500 AGC target 1*10^6 max. Injection time [msec] 400 Isolation window [Da] 0.4 normalized collision energy [instrument units] 40-60 steeped Table S4. Concentrations of total As and all detected As species in the samples, in [mg kg -1 dm].
ID ASP-017 ASP-023 ASP-068 STM-107 STM-108 STM-109
R. R. R. R. cf. R. cf. Species R. cf. Pallida subbotrytis subbotrytis subbotrytis Largentii Pallida Czech Czech Origin Slovakia Austria Austria Austria Republic Republic
total As 25 ± 2 61 ± 5 44 ± 4 1.7 ± 0.1 11.7 ± 0.2 8.3 ± 0.3
extracted 22 ± 2 66 ± 4 39 ± 1 1.4 ± 0.1 9.5 ± 0.1 6.9 ± 0.2 As 0.018 ± 0.005 ± MA 0.09 ± 0.06 0.08 ± 0.01 0.004 ± 0.001 0.007 ± 0.001 0.006 0.002 0.024 ± TMAO 0.42 ± 0.07 0.4 ± 0.03 0.13 ± 0.02 0.009 ± 0.001 0.04 ± 0.01 0.005 0.007 ± 0.007 ± traces traces 0.008 ± As (V) 0.03 ± 0.02 0.002 0.006 (< 0.003) (< 0.003) 0.002
DMA 0.06 ± 0.02 0.094 ± 0.01 0.11 ± 0.01 0.078 ± 0.02 0.11 ± 0.01 0.08 ± 0.01
0.020 ± TETRA 0.05 ± 0.01 0.4 ± 0.02 0.22 ± 0.01 0.04 ± 0.01 0.025 ± 0.002 0.005 traces 0.015 ± DMAA 0.04 ± 0.02 0.06 ± 0.02 0.11 ± 0.02 0.017 ± 0.001 (< 0.003) 0.001
AB 19.8 ± 0.2 60 ± 6 31 ± 7 0.94 ± 0.02 8.4 ± 0.6 5.9 ± 0.1
AC 0.35 ± 0.06 1.8 ± 0.2 0.68 ± 0.06 0.06 ± 0.01 0.15 ± 0.01 0.17 ± 0.02
0.057 ± TMAP 0.03 ± 0.01 0.22 ± 0.06 0.23 ± 0.06 0.018 ± 0.003 0.044 ± 0.002 0.001 0.058 ± 0.064 ± AC2 0.31 ± 0.03 0.18 ± 0.01 0.011 ± 0.001 0.035 ± 0.003 0.006 0.004 unknown 0.079 ± As 0.41 ± 0.08 0.9 ± 0.3 2 ± 1 0.019 ± 0.004 0.099 ± 0.004 0.006 species Synthesis of (3-hydroxypropyl)trimethylarsonium bromide (AC2):
To a 20 mL Pyrex vial (Biotage) equipped with a stirring bar, flushed with argon, and sealed with Teflon septum and aluminum crimp, trimethylarsine (TMAs, 1.8 mL, 16.8 mmol, 1 equiv.) was added via a syringe, followed by 3-bromo-1-propanol (1.5 mL, 17.2 mmol, 1.02 equiv.). The neat reaction mixture was then stirred at 75 °C for 3 days. To the processed reaction mixture, cold acetonitrile was added (10 mL) and the obtained solid was quickly filtered (suction filtration) and washed with additional 10 mL of cold acetonitrile. The product was dried in a desiccator under vacuum for 3 hours to provide (3-hydroxypropyl)trimethylarsonium bromide (AC2 ) as a white solid. Yield: 1.88 g, 43%. NMR data of the synthesized (3-hydroxypropyl)trimethylarsonium bromide:
1 H NMR (300 MHz, D 2O): δ = 3.67 (t, J = 6.0 Hz, 2H), 2.43-2.38 (m, 2H), 1.93-1.83 (m, signal overlapping with Me, 2H), 1.87 (s, 3 x Me, 9H);
13 C NMR (75 MHz, D 2O): δ = 61.0, 24.6, 21.4, 6.5; Conclusion and outlook
5 Conclusion and outlook
Within this thesis, the arsenic concentration and speciation was investigated in different macrofungal samples. The “edible” ink stain bolete Cyanoboletus pulverulentus was identified as arsenic hyperaccumulating mushroom, and it was estimated that chronic consumption of this species can lead to an increased health risk, because almost all of the total arsenic in samples of this mushroom was present as the possibly carcinogenic DMA.
The deer truffles of the genus Elaphomyces also contained very high arsenic concentrations. They were found to be one of the few fungal species that contain MA as major arsenic species. In addition, unusually high concentrations of TMAO and significant concentrations of the labile MA(III) were detected. The latter compound has never been described in mushrooms before, and is also rarely documented in other natural samples.
In the coral mushrooms of the genus Ramaria , two arsenicals that have only been found in the marine biota so far were detected: DMAA and TMAP. The compound AC2 was identified and described for the first time in nature. It could be an important intermediate in the geobiochemical cycle of arsenic.
The results represent very well the huge diversity of arsenic in mushrooms. First, the concentrations can vary immensely between different fungal species. The study on samples of the genus Elaphomyces demonstrates that already within one genus, different species can vary immensely in their accumulation abilities. The samples of E. asperulus contained much lower arsenic concentrations than the samples of the other two Elaphomyces species, and also significantly lower concentrations of alkali elements like potassium or sodium. What’s more , the arsenic concentrations can even differ to a large extent between samples of the same fungal species, as is the case for C. pulverulentus (2.4 – 1300 mg As/kg dm). The arsenic concentration in the fruit-body is independent of the soil arsenic, at least in C. pulverulentus, but very likely in many other macrofungi as well. Consequently, it is possible that more fungal species will be identified as arsenic hyperaccumulators in the future. It remains unclear why certain fungal species accumulate arsenic much more than others, and why arsenic concentrations can span over several orders of magnitude within one species. The identification of influencing factors will be an interesting future task.
The extremely high arsenic concentrations in some samples of C. pulverulentus once again illustrate the importance of arsenic speciation analysis for human health. It is highly recommended to determine the arsenic speciation of all mushrooms that are classified as edible and contain (constantly or even only occasionally) high concentrations of arsenic. It is worth mentioning that the most popular mushroom species, Cantharellus cibarius (Chanterelle, in German: Pfifferling, Eierschwammerl) and
92 Conclusion and outlook
Boletus edulis (Porcino, in German: Steinpilz), contain very low arsenic concentrations. Therefore, there is little risk of being poisoned by arsenic from mushrooms for the vast majority of people.
It was demonstrated that the arsenic speciation of mushrooms can consist of one species alone (DMA in C. pulverulentus ) or of one dominating species and many species at low relative abundances (Ramaria spp. with AB as main arsenical). There are also cases where there is not only one dominating arsenic compound, but also a second species in high relative concentrations (up to 37 % TMAO next to the main arsenical MA in Elaphomyces spp.). Concerning the samples of Elaphomyces, it is fascinating that the extraction efficiency of E. asperulus was very low (only 3 – 14 %), while it was around 80 % in E. granulatus and E. muricatus . This implies that most of the arsenic of E. asperulus is present in the fruit-bodies in a quite complex form, which is not easily extractable with water. This could be for example lipid soluble arsenicals or alternatively large biomolecules, like proteins, where the arsenic is more or less strongly attached. Elucidating these arsenic-containing compounds will be a demanding task, but at the same time a significant contribution for further understanding the dynamics of arsenic in macrofungi.
Another remarkable aspect of the Elaphomcyes samples is their arsenic speciation profile in aqueous extracts. The arsenic compounds that are usually dominant in macrofungi, namely AB, DMA and inorganic arsenic, are only present at trace concentrations in the deer truffles. At the same time, they contain high concentrations of MA and TMAO, and also significant concentrations of MA(III). This leads to speculations whether the arsenic metabolism of Elaphomyces (if an active arsenic metabolism by macrofungi exists) is perhaps more closely related to the arsenic metabolic pathway suggested by Challenger for the formation of DMA and TMAO (which usually assigned to the arsenic metabolism of mammals, at least until the stage of DMA) than to the marine pathways for the formation of AB. However, in this case, the low abundance of DMA as intermediate between MA and TMAO is puzzling. One solution would be that only trivalent DMA(III) is formed, which is extremely unstable and will therefore hardly be found in any natural sample. It will be helpful to investigate other hypogeous macrofungi and compare their arsenic speciation with the results of the samples of Elaphomyces . Another appealing task will be the investigation of Tolypocladium ophioglossoides (already mentioned in the introduction) , which is a parasitic macrofungal species that grows on Elaphomyces fruit-bodies. Preliminary results indicated that the major arsenic species is an unknown compound, which makes the outlook even more exciting. A thorough study of the arsenic speciation in this parasite and at the interface between host and parasite could lead to important insights about the actual site(s) of arsenic biotransformation in the fungal environment, which is still a mystery within the field of arsenic research.
93 Conclusion and outlook
Further, the presence of DMAA, TMAP and the newly identified AC2 in Ramaria samples contributes to the elucidation of the transformation mechanisms, no matter where they actually take place. Since their detection in samples of coral mushrooms, DMAA and TMAP were positively identified in several other macrofungi (data not published), and it is very likely that AC2 will be found in other samples as well. To finally understand the geobiochemical cycling of arsenic, it will be crucial to identify further intermediate species in mushrooms. Logical candidates are on the one hand some aldehydes, for example AB aldehyde or TMAP aldehyde, and on the other hand trimethylated arsenosugars as precursors of AC. These compounds would fit at least to some extent with the proposed pathways for the formation of AB in the marine environment. Of course, it is also possible that the formation of AB in macrofungi (or the adjacent soil) is completely different from the pathways in the marine biota. Again, detection of more arsenic species will be a key step to investigate this further. In vitro experiments will also be crucial, but they have to be planned very carefully, and the transfer of findings from in vitro experiments to the real world is often tricky.
It has to be noted that the discovery of AC2, DMAA and TMAP in Ramaria samples is a good demonstration of the importance of looking not only at the major arsenic species, but also at the lower concentrated ones. Here, the excellent detection limits of HPLC-ICPMS are very helpful, especially with the introduction of CO 2 as optional gas for active use of the carbon enhancement effect and oxygen as reaction gas in the collision/reaction cell of a triple quadrupole setup.
In conclusion, the examples of this thesis reveal how much there is still to be discovered about arsenic dynamics in our environment. We have yet to understand the reason for such immense arsenic concentrations in some macrofungi; the concentrations in C. pulverulentus account for up to 0.1 % dm, which is much more than essential elements like iron and is in the same order of magnitude as sodium or zinc! This is an indication that arsenic could in fact be beneficial for these mushrooms. An actual proof still has to be found.
It is also fascinating that the arsenic speciation can vary so much within different fungal species. The reason for this, as well as the source of the different arsenic species in mushrooms still has to be elucidated. The discovery of new arsenic compounds in mushrooms will be a key step for further investigations on arsenic biotransformation mechanisms.
94 Conclusion and outlook
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