The risk of high mercury accumulation in edible mushrooms cultivated on contaminated substrates

Accepted Manuscript Title: The risk of high mercury accumulation in edible mushrooms cultivated on contaminated substrates Author: Piotr Rzymski Mirosław Mleczek Marek Siwulski Monika G˛asecka Przemysław Niedzielski PII: DOI: Reference:

S0889-1575(16)30082-5 http://dx.doi.org/doi:10.1016/j.jfca.2016.06.009 YJFCA 2734

To appear in: Received date: Revised date: Accepted date:

28-3-2016 28-5-2016 18-6-2016

Please cite this article as: Rzymski, Piotr., Mleczek, Mirosław., Siwulski, Marek., G˛asecka, Monika., & Niedzielski, Przemysław., The risk of high mercury accumulation in edible mushrooms cultivated on contaminated substrates.Journal of Food Composition and Analysis http://dx.doi.org/10.1016/j.jfca.2016.06.009 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Type of paper: Original Research Article

The risk of high mercury accumulation in edible mushrooms cultivated on contaminated substrates Piotr Rzymski1*, Mirosław Mleczek2, Marek Siwulski3, Monika Gąsecka2, Przemysław Niedzielski4 1

Poznan University of Medical Sciences, Department of Environmental Medicine, Poznań,

Poland 2

Poznan University of Life Sciences, Department of Chemistry, Poznań, Poland

3

Poznan University of Life Sciences, Department of Vegetable Crops, Poznań, Poland

4

Adam Mickiewicz University in Poznań, Faculty of Chemistry, Poznań, Poland

* Corresponding author: Piotr Rzymski, [email protected]

1

Highlights

1. The accumulation of Hg in commercially cultivated mushrooms was studied 2. A coincident increase of Hg content in substrate and mushroom bodies was observed 3. The presence of Hg did not alter the carbohydrate, protein and fat content 4. To ensure health safety, substrates should contain the lowest possible Hg content

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Abstract Mercury (Hg) exposures represent a significant worldwide health issue. At the same time its content in cultivated mushrooms is not effectively regulated. The present study investigated how substrate contamination with Hg (0.1-0.5 mM) affects its accumulation in stipes and caps of Agaricus bisporus E58, Pleurotus ostreatus H195 and Hericium erinaceus HE01, mushroom growth and composition of macronutrients. The greatest Hg accumulation was demonstrated for caps. Generally, Hg uptake increased in a concentration-dependent manner and exceeded 44 mg kg-1 (P. ostreatus), 116 mg kg-1 (A. bisporus) and 53 mg kg-1 (H. ercinaceus) in caps after 0.5 mM was added to the substrate. Importantly, an increase in Hg accumulation was also significant and potentially hazardous for human health at the lowest assayed concentration. A. bisporus and P. ostreatus revealed high resistance to Hg and declined its biomass only at 0.4 and 0.5 mM concentration. The presence of Hg did not alter the macronutrient composition (total carbohydrates, proteins and fats). These results highlight the significant role of proper substrate selection in mushroom cultivation to avoid exposing consumers to harmful Hg levels and further health consequences.

Keywords: mercury contamination; food safety; cultivated mushrooms; Pleurotus; Agaricus; Hericium; human health; food composition; food analysis

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1. Introduction Mercury (Hg) is a widely recognized environmental persistent pollutant and a highly toxic element. Its health risks depend on form, concentration, duration and route of exposure (Bernhoft, 2012). However acute toxicity effects of Hg, such as in the Minamata case (Kondo, 2000), may occur rarely and be only limited to particular groups at higher occupational risk (Rajaee et al., 2015), a significant part of the human population may still be chronically exposed to low but still hazardous concentrations of this element (Buchanan et al., 2015). Long-term Hg exposures can lead to toxic effects on cardiovascular, pulmonary, urinary, gastrointestinal, neurological and reproductive levels (Fernandes Azevedo et al., 2012; Rice et al., 2014; Rzymski et al., 2015a). Over the years numerous concerns regarding the increased presence of Hg in various food products such as rice or fish have been raised (Li et al., 2014; Jallad, 2015; Rzymski et al., 2015b) and in response, strict limits in different regions have been implemented (Commission Regulation (EC) No 1881/2006). Nevertheless, Hg contamination still represents a significant environmental issue (Li et al., 2012) underlining a continuous need to identify all routes through which human Hg exposures can occur. Mushrooms are an important part of diet in many world regions (Valverde et al., 2015). Wild edible species collected for culinary reasons have already been found to uptake several toxic elements easily (including Hg) from the ambient environment and accumulate them in aboveground parts (Falandysz et al., 2015; Mleczek et al., 2015a,b). Therefore, the place from which they are collected is crucial if the specimen is to be safe for human health. Cultivation of mushrooms, currently associated with a multi-billion dollar worldwide business (Li et al., 2013), can, in turn, deliver final food products of standardized high-quality and safety only if the specimen are grown on substrates free of contaminants. Our previous study has clearly demonstrated that if the substrates are contaminated even with a low content of arsenic, some species (e.g. Pleurotus ostreatus and P. eryngii) may accumulate its high levels and pose a threat to human health . There is a possibility that a similar phenomenon could also occur in relation to Hg contamination but only a few non-comparative studies on this matter have been performed so far (Enke et al., 1979; Bressa et al., 1988). Typical substrates used to cultivate mushrooms include wheat straw, gypsum, oak and beech sawdust, or chicken manure (Sánchez, 2010; Niedzielski et al., 2014; Mleczek et al., 2015c). All of the above can potentially contain increased Hg concentration, particularly if collected from polluted areas (Baloch, 1999). 4

The present study aimed to investigate experimentally to what extent the contamination of substrate with Hg can influence its accumulation in the edible parts of some of the most popular mushroom species in the world: buttom mushroom Agaricus bisporus (J.E. Lange) Imbach), oyster mushroom Pleurotus ostreatus (Jacq.) P. Kumm.) and lion’s mane mushroom Hericium erinaceus (Bull.:Fr.) Pers. Moreover, the effects of Hg on the biomass of these mushrooms and their macronutrient composition were also studied. The findings of this study are important in view of the safety of final food products obtained from the investigated mushroom species. 2. Materials and methods 2.1. Experiment design The methods of mushrooms cultivation described in this experiment were similar to that commonly used in commercial cultivation (Oei, 2003, Sokół et al., 2016). Agaricus bisporus In the experiment the pasteurized compost from commercial production of button mushroom was used. The compost was prepared from a mixture of wheat straw and poultry manure following a conventional method of fermentation and pasteurization. The Hg content in substrate was 0.037±0.012 mg kg-1. The compost was mixed with mercury(II) nitrate (Hg(NO3)2 H2O) (Sigma-Aldrich, Saint Louis, USA) solution to a final concentration of 0.1, 0.2, 0.3, 0.4 and 0.5 mM. The compost was then mixed with grain mycelium (on wheat grain) of Sylvan A15 strain (cultivated in Poland) using a POLYMIX PX-SR 90 D stirrer (KINEMATICA AG, Littau-Luzern, Switzerland) in the amount of 5% of the substrate mass and place in plastic containers (13 x 25 x 35 cm). Each of the contained 3 kg of spawned compost. Incubation was carried out in the dark at 25±1°C and 95±2% air humidity until the compost became overgrown by mycelium. Next, the compost was covered with a 5 cm layer of casing soil (with Hg content below limit of detection)The casing soil was prepared from mixture of black peat and chalk (20 g of chalk per 10 dm3 of peat). The mixture was watered to 70% moisture content. After compost covering, the incubation was conducted in the above mentioned conditions. Once the casing soil became overgrown by mycelium, the temperature was decreased to 17°C. The casing soil was watered to maintain constant moisture. The cultivation chamber was aerated to keep the CO2 concentration below 1000 ppm and air humidity at 85-90%. Whole carpophores of the first flush of fructification were harvested. Three independent experimental replications were conducted. Pleurotus ostreatus

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The substrate for P. ostreatus was prepared from wheat straw cut into chaff 4-5 cm long. The Hg content in substrate was below the detection limit. The straw for the experiment was moistened to 60%, packed into plastic bags and pasteurized at 60ºC for 24 hours. The same concentration range of Hg as described earlier was used. The final moisture content was similar to that in A. bisporus experiments. The pasteurized straw was then mixed with grain mycelium (on wheat grain) H195 strain (cultivated in Poland) which constituted 3% in relation to the wet weight of the substrate. The substrate was placed in bags of perforated polyethylene foil at the amount of 1 kg per bag. The incubation was carried out at 25±1°C and 90±2% air humidity. When the substrate was totally overgrown by mycelium, it was transferred to the cultivation chamber. The cultivation was conducted at the 15-16°C and 8590% of air humidity. Fluorescent light irradiation of 500 lx was also used with a photoperiod of 10 h light and 14 h darkness. The CO2 concentration level was maintained below 1000 ppm by controlled aeration. Carpophores with stipe were harvested as they matured. Three independent experimental replications were conducted. Hericium erinaceus The substrate for the H. erinaceus HE01 strain (cultivated in Poland) was prepared from a mixture of beech and oak sawdust (3:1 vol.) which was additionally supplemented with 20% wheat bran, 5% corn flour and 1% gypsum. The Hg content in substrate was 0.089±0.021 mg kg-1. The mixture was watered with distilled water to 45% of moisture. The substrate was placed in polypropylene bags and sterilized at 121°C for 1 h and then cooled down to 25°C. The same concentration range of Hg as described earlier was used. The final moisture of the substrate was 60%. The wet substrate was mixed with grain spawn (on wheat grain) at the amount of 5% of substrate weight and placed in 1 dm3 polypropylene bottles. Each bottle was filled with 350 g of the substrate and closed with a lid. The lid contained a cotton wool filter allowing the exchange of gas. The incubation was carried at 25 ±1°C and 80-85% of air humidity until the substrate was overgrown by mycelium. Next, the bottles with removed lids were placed in the cultivation chamber at a temperature of 16±1ºC and 85-90% of air humidity. An irradiance of 500 lx fluorescent light with a photoperiod of 12 h light and 12 h darkness was applied. Whole carpophores were harvested as they matured. Three independent experimental replications were conducted. 2.2. Chemicals Water has been cleaned by ultrapurification system Millipore (USA) to ultratrace purity. All reagents were an analytical purity grade. For sample preparation, the concentrated (65%) nitric acid (Suprapure, Merck Germany) has been used. The mercury commercial standard 6

100 mg L-1 (Romil, GB) has been used after appropriate dilution. The palladium (as nitrate) modifier solution (Merck, Germany) has been used as a matrix modifier. 2.3. Mushroom sample preparation Collected mushrooms were dried (separately caps and stipes) using an electric oven (SLW 53 STD, Pol-Eko, Poland) at 60±1°C for 48 h with calculation of dry mass. In the case of H. erinaceus, there is no concrete stipe organ, therefore we analysed part of the fruiting bodies directly adjoining the carpohore head and named this part "stipes". Dried materials were ground for 2 min in a Cutting Boll Mill PM 200 (Retsch GmbH, Haan Germany). Three representative samples (0.5000±0.0001 g) for each mushroom species and each Hg addition were mineralized in a closed microwave mineralization system, CEM Mars 5 Xpress (CEM Corp., Matthews, NC, USA) using 8 mL of 65% HNO3 (Sigma-Aldrich) and 1 mL of 30% H2O2 (Sigma-Aldrich) in 55 mL vessels. Digestion of the mushroom samples was performed according to a three stage microwave programme: first stage – power 800 W, temperature 80°C, time 5 min; second stage – power 800 W, temperature 100°C, time 5 min; and the third stage – power 1200 W, temperature 200°C, time 10 min. All materials after digestion were filtered through 45-mm filters (Qualitative Filter Papers Whatman, Grade 595: 4-7 µm) and finally were made up to a final volume of 50 mL with water (Millipore, Saint Louis, USA). 2. 4. Determination of Hg in bodies Total Hg concentration was measured using electrothermal atomic absorption spectrometry (ETAAS) with Zeeman background correction in all three mushroom species (Sardans et al., 2010). A SpectrAA 280Z (Agilent Technologies, Mulgrave, Victoria, Australia) instrument with pyrolytic graphite tubes and an Hg lamp (wavelength 253.7 nm, slit 0.5 nm, current 4 mA) was used. The temperature programme was optimized: drying 85120°C for 55 s; ashing 400°C for 8 s; atomization 1800°C. Palladium solution (10 µL of 1000 mg L-1 for 20 µL of sample) was used as a chemical modifier. The limit of detection, 0.01 mg kg-1 and the uncertainty of results (measured as RSD) at a level of 5.0% were obtained. Traceability was measured by a standard addition procedure. 2.5. Analysis of chemical composition of mushroom The crude ash, fat, protein and carbohydrates were determined according to AOAC standard methods (AOAC 2012). For the crude protein calculation N×4.38 factor was used. Energy value of mushrooms was calculated by multiplying their content of crude protein, fat, and carbohydrate by the Atwater Factors (FAO, 2003). 2.6. Statistical analysis

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Statistical analyses were performed using Statistica 10.0 (StatSoft, USA) and consisted of ANOVA followed by post hoc Tukey’s test. p < 0.05 was considered as statistically significant. 3. Results 3.1. Content of Hg in mushroom species Generally, the content of Hg in the caps and stipes of all tested mushroom species increased over its initial substrate concentration. Moreover, a significant increase in Hg accumulation in all three species was noted at the lowest assayed concentration (Tab. 1). Significant positive and strong correlations between initial substrate content and that accumulated in the caps and stipes of P. ostreatus, A bisporus and H. erinaceus were found and ranged from 0.97 to 0.99. Regardless of the initial concentrations and mushroom species, Hg was accumulated predominately in caps. At low Hg substrate concentrations (0.1 and 0.2 mM), the greatest cap accumulation was found for H. erinaceus while at higher Hg levels (≥0.3 mM) for A. bisporus (Tab. 1). 3.2. Biomass and macronutrient composition The response of mushrooms to substrate Hg concentration was measured by the means of their biomass (Fig. 1). In the case of P. ostreatus, the biomass exhibited significant decrease only at the highest assayed Hg concentration. A. bisporus, in turn, revealed the greatest biomass decrease at 0.4 and 0.5 mM while at the lowest assayed concentration (0.1 mM), an increase compared to control was observed. The greatest adverse effect of Hg presence in substrate was observed for H. erinaceus. The biomass of this species decreased over the substrate Hg concentration with significant changes noted from 0.2 mM and no biomass found once the substrate was supplemented with 0.5 mM of Hg (Fig. 1). Importantly, the aboveground parts revealed no visible toxicity symptoms of Hg uptake (Fig. 2). Hg-induced alterations in biomass of mushrooms were not accompanied by statistically significant changes in macronutrient composition measured as a percentage of carbohydrates, crude fat, proteins and ash. No changes in moisture were noted (Tab. 2).

4. Discussion The worldwide consumption of mushrooms has increased at a relatively high rate and now exceeds 4 kg per person annually compared to only 1 kg in 1997 (Royse, 2014). Various populations have a long tradition of collecting and consuming wild edible mushrooms but undoubtedly the cultivated species are also increasingly consumed in various geographical regions including the US, Asia or Europe (Peintner et al., 2013). Their global production has 8

been developing progressively over recent decades from 1 billion kg in 1978 to nearly 27 million tons in 2012 (Royse, 2014). The most popular cultivated mushrooms species include those from the Pleurotus and Agaricus genus (Sánchez, 2010). As a source of carbohydrate, protein, fats and minerals they represent a highly appreciated delicacy, particularly in Asia and Europe (Latiff et al., 1996; Kalač, 2013). For example, the mean annual consumption of A. bisporus in Europe was recently estimated at 2 kg per capita (Jaworska et al., 2015). The growing interest in these species is also supported by their wide-range of potential therapeutic properties (Khan et al., 2013; Kunjadia et al., 2014; Elbatrawy et al., 2015). Under these circumstances it is of high importance to test whether substrate quality has any impact on the quality of the final food product. The present study clearly highlights the role of keeping the Hg levels in cultivation substrate as low as possible due to the possible uptake and accumulation of this toxic agent in consumed aboveground parts, stipes and caps of P. ostreatus, A. bisporus and H. erinaceus, at highly hazardous levels for human health. Considering that the Provisional Tolerable Weekly Intake (PTWI) for Hg was set at 4 µg kg bw (JECFA, 2010), a single consumption of 100 g of mushroom caps cultivated on substrates contaminated with 0.1 mM of Hg by a 70 kg adult would result in exceeding the PTWI by as much as 419% for H. erinaceus (total Hg dose 1.17 mg), 335% for A. bisporus (total Hg dose 0.94 mg), and 186% for P. ostreatus (total Hg dose 0.52 mg). Consumption of these mushrooms on a daily basis would therefore lead to chronic exposure to significant levels of Hg and pose a relevant health threat. Moreover, it has been postulated that some mushroom species may be capable of partial methylation of inorganic Hg which increases the toxicity of this element (Fisher et al., 1995; Rieder et al., 2011). Whether this phenomenon occurs in the investigated mushroom species requires further studies. These findings are very important in view of the increasing consumption of the studied species and the lack of regulations concerning the monitoring of the quality of commercially available cultivated mushrooms in many parts of the world. For example, in the European Union the Hg regulations on foodstuffs only concern seafood and dietary supplements (Commission Regulation (EC) 1881/2006). Moreover, unlike arsenic (Tao & Bolger, 1999; Niedzielski et al., 2013; Melgar et al., 2014), the levels of Hg in commercially available mushroom products in various geographical regions has not been subject to comprehensive investigations. This is surprising given that wild edible species, including those from the Agaricus genus, have

already been shown to uptake and translocate significant Hg

concentrations into their aboveground parts (Kuusi et al., 1981; Falandysz et al., 2003). It was, however, recently demonstrated that A. bisporus mushrooms available on the Chinese 9

market are characterized by the greatest levels of Hg from all ten tested mushroom species Lentinus edodes, Auricularia auricula, P. ostreatus, Tremella fuciformis, Flammulina velutipes, Agrocybe chaxinggu, Armillaria mellea, A. bisporus, and Pholiota nameko (Huang et al., 2015). Given that concentrations of Hg in stipes and caps of cultivated mushrooms resulting from substrate contamination could reach levels highly hazardous to human health, we are of the opinion that not only final mushroom products should be subject to routine monitoring but also substrate quality should be controlled. Another important finding of our study is the effect of Hg substrate contamination on mushroom biomass and its macronutrient composition. As demonstrated, H. erinaceus was susceptible to Hg contamination and its yielded biomass was significantly decreased in the concentration-dependent manner with no yield at 0.5 mM initial substrate content of Hg.. A, bisporus and P. ostreatus, in turn, revealed a significant decrease in biomass only at 0.4 and 0.5 mM or 0.5 mM concentration of Hg, respectively. It is, however, important to notice that the biomass was still relatively high and by means of yield could be regarded as satisfactory and did not reveal any visible symptoms of Hg toxicity within stipe and cap parts. This, in turn, raises the significant risk that Hg-contaminated mushrooms, particularly A. bisporus and P. ostreatus, may not be recognized by cultivators and later, by consumers as potentially hazardous for health. Furthermore, the addition of Hg and the accumulation of this element in caps and stipes did not cause any simultaneous changes in general macronutrient composition as total carbohydrate, protein and fat content in mushrooms of all three tested species did not differ significantly between specimens grown on Hg-free and Hg-contaminated substrates (regardless of the Hg concentration). Consequently, mushrooms representing a significant and hazardous source of this toxic element could still be mistakenly regarded as nutritious and not withdrawn from the market. This again, strongly advocates the need to screen Hg content in commercially-available mushrooms and to establish an upper safety level for this food product on various economic markets. In summary, the present study investigated the response of popular edible mushroom species to Hg presence in overgrown substrate. Not only were significant and potentially hazardous levels of this element uptaken and accumulated in aboveground mushroom parts once the substrate was artificially contaminated with low Hg concentrations but the yielded biomass and composition of macronutrients was not decreased. These results underline the meaningful role of selecting the substrate for mushroom cultivation characterized by the lowest possible Hg concentration to avoid consumer exposure to harmful Hg levels and its further health consequences. Moreover, it seems reasonable to perform screening of Hg 10

content in commercially available cultivated mushroom products introduced to various markets and to set a maximum permissible levels.

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Figure captions Fig 1. Characteristics of tested mushroom biomass [g] Fig. 2. Macroscopic characteristics of mushrooms in relation to Hg addition to substrate

16

17

Fig 1. Characteristics of tested mushroom biomass [g]

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Fig. 2. Macroscopic characteristics of mushrooms in relation to Hg addition to substrate

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Table 1. Content (mean±SD) of Hg [mg kg-1 DW] in fruiting bodies of tested mushroom species after substrate supplementation with different Hg addition. Hg

P. ostreatus H195

A. bisporus E58

H. erinaceus HE01

addition [mM]

cap

stipe

cap

stipe

cap

stipe

control

0.10±0.01f

0.02±0.01e

0.08±0.01f

0.05±0.01e

0.07±0.02f

0.03±0.01e

0.1

5.2±0.2e

2.4±0.2d

9.4±0.7e

4.1±1.0d

11.74±1.2e

6.0±0.8d

0.2

23.6±1.5d

13.7±1.0c

25.1±1.4d

18.2±1.2c

27.19±2.5d

10.4±0.8c

0.3

35.8±1.6c

26.0±1.4b

40.9±2.0c

20.7±4.1c

38.09±2.3c

15.6±1.5b

0.4

41.0±1.3b

27.2±1.2ab

89.1±3.8b

48.9±2.5b

51.62±1.8b

22.7±1.8a

0.5

44.6±1.7a

29.5±1.3a

116.4±7.2a

53.6±1.6a

53.99±2.2a

23.3±2.1a

Mean values (n=3) ± standard deviations; identical superscripts denote no significant difference (p > 0.05) between mean values in columns according to Tukey's HSD test (MANOVA) for caps and stalks of particular mushroom species

20

Table 2. Chemical composition (mean±SD) of tested mushroom species cultivated on substrates supplemented with Hg. No significant difference (p > 0.05) between mean values in columns according to Tukey's HSD test (MANOVA) were found. Hg addition

P. ostreatus

A. bisporus

H. erinaceus

[mM]

H195

E58

HE01

Carbohydrates [%] control

40 ± 1

34 ± 2

44 ± 1

0.1

41 ± 2

35 ± 2

42 ± 1

0.2

41 ± 2

34 ± 2

43 ± 1

0.3

41 ± 2

34 ± 2

42 ± 2

0.4

42 ± 2

35 ± 1

42 ± 1

0.5

42 ± 2

37 ± 2

nb

Crude fat [%] control

2.4±0.3

1.0±0.3

1.3±0.3

0.1

2.0±0.3

1.1±0.2

1.2±0.3

0.2

2.2±0.2

1.1±0.5

1.2±0.5

0.3

2.5±0.5

0.9±0.3

1.6±0.3

0.4

2.3±0.5

1.1±0.5

1.3±0.3

0.5

2.3±0.3

0.8±0.2

nb

Crude protein [%] control

27 ± 2

22 ± 3

21 ± 2

0.1

26 ± 3

22 ± 2

20 ± 1

0.2

26 ± 4

22 ± 1

20 ± 1

0.3

26 ± 1

21 ± 1

20 ± 1

0.4

26 ± 1

20 ± 1

19 ± 1

0.5

25 ± 1

20 ± 1

nb

Crude ash [%] control

5.9±0.1

9.4±0.3

8.8±0.1

0.1

6.0±0.2

9.4±0.1

8.7±0.2

0.2

5.9±0.2

9.4±0.3

8.7±0.2

0.3

6.0±0.1

9.4±0.1

8.9±0.2 21

0.4

6.2±0.2

9.2±0.3

8.7±0.1

0.5

6.1±0.2

9.2±0.2

nb

Moisture [%] control

91.4±0.2

91.9±0.3

88.9±0.4

0.1

91.4±0.3

91.8±0.4

88.7±0.5

0.2

91.4±0.2

91.9±0.2

89.5±0.4

0.3

91.4±0.2

92.0±0.3

88.8±0.4

0.4

91.5±0.3

92.0±0.3

88.5±0.6

0.5

91.4±0.2

91.8±0.2

nb

Energy value [kcal/g] control

2.9

2.4

2.7

0.1

2.9

2.4

2.6

0.2

2.9

2.3

2.6

0.3

2.9

2.3

2.6

0.4

2.9

2.3

2.5

0.5

2.9

2.3

nb

Mean values (n=3) ± standard deviations; nb – no bodies

22