Article pubs.acs.org/est
Arsenic, Lead, and Cadmium in U.S. Mushrooms and Substrate in Relation to Dietary Exposure Angelia L. Seyfferth,* Colleen McClatchy,‡ and Michelle Paukett§ Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware 19716, United States S Supporting Information *
ABSTRACT: Wild mushrooms can absorb high quantities of metal(loid)s, yet the concentration, speciation, and localization of As, Pb, and Cd in cultivated mushrooms, particularly in the United States, are unresolved. We collected 40 samples of 12 types of raw mushrooms from 2 geographic locations that produce the majority of marketable U.S. mushrooms and analyzed the total As, Pb, and Cd content, the speciation and localization of As in select samples, and assessed the metal sources and substrate-to-fruit transfer at one representative farm. Cremini mushrooms contained significantly higher total As concentrations than Shiitake and localized the As differently; while As in Cremini was distributed throughout the fruiting body, it was localized to the hymenophore region in Shiitake. Cd was significantly higher in Royal Trumpet than in White Button, Cremini, and Portobello, while no difference was observed in Pb levels among the mushrooms. Concentrations of As, Pb, and Cd were less than 1 μg g−1 d.w. in all mushroom samples, and the overall risk of As, Cd, and Pb intake from mushroom consumption is low in the U.S. However, higher percentages of tolerable intake levels are observed when calculating risk based on single serving-sizes or when substrate contains elevated levels of metal(loid)s.
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INTRODUCTION
Most of the research on metal(loid)s in mushrooms conducted to date has focused on wild mushrooms growing near contaminated areas in Europe, which can be used as indicators of pollution,16 with relatively less attention given to cultivated mushrooms. Both inedible and edible wild mushrooms have long been known to tolerate and accumulate high concentrations of metal(loid)s depending on the particular metal(loid), the substrate, the mushroom species, and the age of the mycelium.17−20 Mushrooms growing on contaminated soil or near mining areas contain elevated levels of As, Cd, Hg, Pb with bioaccumulation factors (BAF) generally very low for Pb (1), and highly dependent on mushroom species and substrate concentration for As (0.03− 38); BAFs decrease as substrate concentration increases for Cd and As, indicating a potential regulatory mechanism.17,20−29 While relatively few studies have focused on health risks of cultivated mushrooms grown in noncontaminated areas, recent market surveys from noncontaminated areas have shown that cultivated mushroom consumption in China,30 Spain,31 and Canada32 poses little health risk. Cultivation practices for a majority of commonly grown mushrooms in the U.S. (i.e., Agaricus bisporus, cv. White Button, Cremini, Portobello) differ from other parts of the world and may impact levels of metal(loid)s such as Pb, Cd, and As in the mushroom fruit. For example, poultry litter, which may contain elevated As
The transfer of toxic trace metal(loid)s from soil into the human diet may pose health risks; thus, it is important to understand not only the concentration but also the speciation and localization of these elements in foodstuffs. Inorganic As, Pb, and Cd are among the most highly toxic inorganic trace elements present in soils and are detected in foodstuffs worldwide, yet each has different modes of mobility in soils, uptake into produce, and toxicity to humans. Chronic Pb exposure causes toxicity to every human organ system most notably the renal, reproductive, and nervous systems; children are particularly sensitive to Pb exposure and suffer neurological problems at Pb levels much lower than in adults.1 Cd is not easily excreted from the body and thus accumulates over a lifetime causing kidney damage and skeletal abnormalities.2 Chronic exposure to inorganic As (henceforth denoted Asi) leads to skin, bladder, and lung cancers.3,4 In the United States, Cd and Asi ingestion in nonsmokers occurs mainly through foodstuffs whereby crop plants are effective at translocating these toxins from soil solution through roots to the edible portions under certain biogeochemical conditions.3,5−14 Pb, however, is ingested mainly through drinking water (Pb pipes) and through foodstuffs when Pb-containing soil particles are poorly washed from fresh produce.15 While extensive work with higher plants have revealed that Pb is generally excluded from shoots and Cd and Asi are more readily translocated to shoots depending on local conditions, relatively less is known about the uptake mechanisms and storage of toxic metal(loid)s into higher fungi and, in particular, cultivated mushrooms. © XXXX American Chemical Society
Received: April 28, 2016 Revised: June 16, 2016 Accepted: August 2, 2016
A
DOI: 10.1021/acs.est.6b02133 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Environmental Science & Technology levels,33,34 is one part of an array of starting materials commonly used to make mushroom compost and little is known about the transfer of As from substrate to mushroom fruit in U.S. mushrooms. A summary of results from the US Food and Drug Administration’s Total Diet Study (TDS) from 1994−2011 shows that the total As concentration in U.S. mushrooms is surprisingly similar to that of white rice (Figure S1), which is considered the primary route of As exposure in the typical U.S. diet.6,8,35,36 While the TDS results suggest that raw mushrooms may contribute to human Asi ingestion, the study did not report chemical speciation of As. It is well-known that a range of toxicities exist among the various As species found in soils and foodstuffs as opposed to Cd and Pb, for which the toxic divalent forms are predominant. Some organic As (Aso) compounds are completely nontoxic (e.g., arsenobetaine (AsB) and arsenocholine), other Aso compounds are fairly toxic (e.g., dimethylarsenous acid (DMA) and monomethylarsinic acid (MMA)) and Asi compounds are the most acutely toxic (arsenite (As(III)i) and arsenate (As(V)i)).37,38 Slejkovec et al.27 analyzed the As speciation in over 50 species of mushrooms collected from Slovenia, Switzerland, Brazil, The Netherlands, Sweden, and in one sample from the U.S. and found variations in As species among mushroom species. While the TDS provides a starting point to indicate that mushroom consumption may contribute to human Asi, Pb, or Cd ingestion, the mechanistic pathway of metal(loid) transfer into these tested mushrooms, the speciation and localization of As within the mushrooms, the type of mushroom tested, and potential human-health risk in U.S. mushrooms remain elusive. Here, we evaluated the human-health risk associated with ingestion of Asi, Pb, and Cd via raw mushrooms in the United States, identified the localization of As within two common mushroom types, and elucidated the source of substrate-tomushroom transfer. We hypothesized that (1) Asi concentrations will vary with mushroom type and geographic location and (2) the source of As, Cd, and Pb to mushrooms involves compost ingredients such as poultry litter. To test these, we obtained 40 raw mushroom samples consisting of 12 mushroom types from 2 geographic regions that represent most of the cultivated mushroom production in the U.S. We particularly focused our sampling on the most commonly consumed mushrooms in the U.S. and included other specialty mushrooms in our sampling. In addition, we sampled the starting materials from which mushroom compost was prepared, and compost at various stages of preparation, at one mushroom farm to determine the source of As, Cd, and Pb to mushrooms. Total As, Pb, and Cd and As species concentrations were determined on mushrooms and compost ingredients, and the localization of As in select mushrooms was analyzed with synchrotron X-ray fluorescence (XRF) imaging; these data are presented in the context of human health.
were Agaricus bisporus (cv. White Button, n = 11), A. bisporus (cv. Cremini, n = 6), A. bisporus (cv. Portobello, n = 7), Lentinula edodes (cv. Shiitake, n = 5), Pleurotus eryngii (cv. Royal Trumpet, n = 2), P. ostreatus (cv. Oyster, n = 3), Pholiota nameko (cv. Forest Nameko, n = 1), Grifola frondosa (cv. Maitake, n = 1), Flammulina velutipes (cv. Enokitake, n = 1), Hypsizygus tessellatus (cv. Alba Clamshell (Bunapi-Shimeji), n = 1), Hypsizygus tessellatus (cv. Brown Clamshell (Buna-Shimeji), n = 1), and Cantharellus cibarius (cv. Chanterelle, n = 1). Of these, all were collected from six farms in Chester County, PA, except for one sample each of White Button, Portobello, Cremini, Alba Clamshell, Brown Clamshell, Forest Nameko, and Royal Trumpet, which were from three farms in California. For each of the 40 samples, at least four and as many as 10 mushrooms were combined into a composite sample. Substrate and Mushroom Collection. At one mushroom farm in Chester County, PA, additional samples of White Button mushrooms and growth substrate were collected to track As, Pb, and Cd transfer from substrate to fruit. Starting materials for mushroom growth substrate (i.e., compost) were collected and included corn cobs, cottonseed hulls, cocoa hulls, wheat straw, gypsum from recycled dry-wall material, cottonseed meal, peat moss, horse manure, and chicken manure. Compost at different stages of development were also collected and included the “pre-wet”, which is a precompost mix of starting materials, “mid-compost”, which is compost during the composting process, and “final compost” collected at the final stage of compost development. Finally, fully developed white button mushroom fruits, hyphae, and substrate were collected from grow houses at this facility. For each sample, at least 0.5 L volume of material was obtained. Sample Processing, Digestion, and Total Elemental Analysis. Samples were collected into brown paper bags and were brought back to the laboratory. Fresh mushrooms were bisected longitudinally with a stainless steel blade; one-half was archived in a −80 °C freezer and the other half was used for analysis. A subset of mushroom samplesone Cremini and one Shiitake mushroomwere freeze-dried, embedded in epoxy (EPO-TEK 301 2FL) resin, and sectioned after hardening to create a 30 μm-thick section through the middle of the fruit that was utilized for X-ray fluorescence (XRF) imaging; per previous work, this embedding method does not alter As species distributions.39 In addition, a portion of the collected White Button mushroom substrate was dried under anoxic conditions, embedded in epoxy resin and similarly sectioned for XRF imaging. The remaining samples were ovendried at 65 °C and ground into fine powders with stainless steel blades. Powdered mushroom samples were weighed into Teflon digestion vessels (CEM Corporation, Matthews, NC, USA) and were subject to microwave-assisted acid digestion with HNO3 (MARS 6, CEM Corporation) according to previous work.40,41 Total As, Pb, and Cd were analyzed in the HNO3 digest after dilution using ICP-MS (Agilent) with a He collision cell to minimize interference from ArCl+ on m/z 75 for As. Note that while As was analyzed in all samples, Cd and Pb were determined only on mushroom samples used for As speciation and for all mushroom samples in the substrate-tomushroom survey. Quality assurance and quality control was assured by using spike recoveries and standards checks and blanks with analytical runs. Using NIST Certified Reference Material (CRM) 1568a rice flour, As recovery was 103 ± 6% (n = 3) of the certified value, and using WEPAL 883 Carnation
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EXPERIMENTAL SECTION Mushroom Market Survey. A total of 40 raw mushroom samples consisting of 12 mushroom types were collected in summer 2013 from farms in Chester County, PA (n = 33), and a supermarket in Menlo Park, CA (n = 7), which together represent geographic locations from which well over half of the U.S. mushrooms are grown. We focused our sampling on the most commonly consumed mushrooms in the U.S. and also include several specialty mushroom samples that were available at the sampling locations. The 12 mushroom types sampled B
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Figure 1. Total dry weight concentrations of total As (A), inorganic As (B), Pb (C), and Cd (D) found in raw mushrooms in the U.S. in this study. Different letters above the data indicate significant differences with Duncan’s multiple comparison test at least at the 10% level. Mushrooms containing As higher than that depicted by the dotted line (ca. 0.4 μg g−1) in A were used in speciation analyses. The dashed line in B−C represents the 1% level of tolerable Asi and Pb, and the 0.1% level of tolerable Cd ingestion via consumption of mushrooms based on average consumption rates. ○ White Button; • Cremini; □ Portobello; ■ Shiitake; △ Royal Trumpet; ▲ Oyster; ◊ Forest Nameko; ⧫ Maitake; × Enoki; + Alba Clamshell; □ Brown Clamshell; ☆ Chanterelle.
on Shiitake utilizing a pixel size of 2 μm and dwell time of 50 ms per pixel on beamline 2-3, which is optimized for microfocused XRF imaging. On both beamlines, the samples were mounted onto an aluminum sample holder placed at 45° to the incident beam. Samples were imaged utilizing an incident energy of 12 000 eV for mushroom fruit and 13 000 eV for mushroom substrate. Fluorescence intensities of total As, Fe, Ca, Mn, P, S, K, Ti, Cu, and Zn were monitored with a vortex detector placed 45° to the sample (90° to the incident beam) as the sample was rastered in the microbeam with a Newport stage. Because Lα shell emission lines of Pb overlap with Kα shell emission lines of As, Pb was unable to be analyzed, and signal is due to As because the incident energy is lower than the Pb L-edge binding energy. Likewise, Cd was unable to be analyzed because the Lα shell emission lines of Cd overlap with Kα shell emission lines of K and Ca. Element counts were derived by integrating the intensity over appropriate regions of interest (ROIs). Contamination from neighboring elements was minimized by maintaining narrow ROI windows. Background counts were determined by inspecting a histogram of the low signal intensity in nonsample regions and subtracting the counts corresponding to the cumulative counts at one standard deviation of the Gaussian noise-distribution (∼85% of the total background) from the absolute signal intensity. Statistical Analyses. The concentration of total As, Cd, Pb, and Asi were compared among mushroom types for which more than one sample was analyzed per type with ANOVA and Duncan’s multiple comparison’s tests after verifying the data complied to the normality and homogeneity of variance assumptions required of ANOVA using the Shapiro−Wilks and Levene’s tests, respectively. Raw data of total As in mushrooms were not normally distributed, and thus the square root transformation of the data were used to comply to the
straw, Pb and Cd recoveries were 109 and 106%, respectively of the reported value (Table S1). As Speciation. Powdered mushrooms that contained total As higher than 0.4 μg g−1 were subject to methanol/water extraction with sonication, centrifugation, and filtration according to the methods of Smith et al.42 and analyzed within 24 h with HPLC-ICP-MS using a PEEK PRP-X100 anion exchange column (250 mm × 4.6 mm, 10 μm) and with an isocratic 10 mM NH4NO3/10 mM NH4H2PO4 eluent (pH = 9.4) at 1.0 mL min−1 to quantify inorganic As (As(V)i and As(III)i) and organic As (MMA, DMA, and AsB) species per previous work.43 Using this separation method, retention times for AsB, As(III)i, DMA, MMA, and As(V)i were 2.5, 3.1, 3.4, 6.5, and 8.7 min, respectively. While no certified mushroom sample exists, we used rice flour (NIST CRM 1568a) to check accuracy of the speciation, and results for As(III), As(V), DMA, and MMA agreed with previously reported work44 and were 0.086 ± 0.005, 0.037 ± 0.003, 0.152 ± 0.018, 0.011 ± 0.001 mg kg−1, respectively. Note that rice flour does not contain AsB; therefore, spike additions into mushrooms samples were performed and gave recoveries of 87 (±2, n = 2)% for AsB. XRF Imaging. XRF images were obtained on the Cremini and Shiitake mushroom and mushroom compost thin sections (Figure S2) at Stanford Synchrotron Radiation Lightsource (SSRL) beamline 10-2, and the Shiitake mushroom was further analyzed on beamline 2-3. Beamline 10-2 is equipped with a 30 pole, 1.45 T Wiggler ID end station and a double crystal (Si 111) monochromator and is optimized for continuousscanning, large aperture XRF imaging. A pinhole aperture of 20 μm was utilized for imaging with a pixel size and dwell time per pixel of 25 μm and 75 ms for Cremini, 20 μm and 25 ms for Shiitake, and 30 μm and 50 ms for mushroom substrate. On select locations (i.e., hotspots), additional images were obtained C
DOI: 10.1021/acs.est.6b02133 Environ. Sci. Technol. XXXX, XXX, XXX−XXX
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Environmental Science & Technology Table 1. Arsenic Species (d.w.) in Select Mushroom Samples (total As ≥ 0.4 μg g−1) from PA and CAa μg g−1 AsB
MMA
DMA
White Button Forest Nameko
ndb 0.08
nd nd
0.08 0.10
White Button White Button White Button White Button White Button Cremini Cremini Cremini Cremini Maitake Portobello Portobello Portobello Portobello Royal Trumpet
0.12 0.04 nd 0.08 nd 0.05 0.08 0.06 nd 0.05 0.06 0.03 0.01 nd 0.01
nd nd nd nd nd nd nd nd nd nd nd nd 0.01 nd nd
0.23 0.13 0.08 0.10 0.21 0.18 0.16 0.12 0.12 0.07 0.06 nd 0.15
As(V)i
As(III)i
Californian Samples nd 0.25 0.01 0.37 Pennsylvanian Samples nd 0.63 0.01 0.45 nd 0.44 nd 0.26 nd 0.02 nd 0.61 nd 0.58 0.01 0.38 0.05 0.11 0.01 0.30 0.01 0.23 0.01 0.20 0.01 0.20 0.03 0.07 nd 0.42
sum Asi
residual As
%AsB
%DMA
%Asi
0.25 0.38
0.31 nd
nd 14
12 18
39 70
0.63 0.46 0.44 0.26 0.02 0.61 0.58 0.39 0.16 0.31 0.24 0.21 0.21 0.10 0.42
0.02 nd nd nd 0.42 nd 0.06 nd 0.28 nd 0.09 0.19 0.20 0.30 nd
12 7
23 23 17 25 nd 24 20 28 nd 28 24 14 12 nd 28
63 78 94 62 5 70 65 69 37 70 47 42 42 25 78
20 nd 6 9 11 nd 11 12 6 2 nd 2
a
AsB = arsenobetaine, MMA = monomethylarsenous acid, DMA = dimethylarsinic acid, As(V)i = arsenate, As(III)i = arsenite, residual As = total As − ∑AsB, MMA, DMA, Asi. bNot detected.
Figure 2. X-ray fluorescence images showing localization of Ca (A, C) and As (B, D) in 30-μm-thin sections of Shiitake (A, B) and Cremini (C, D) mushroom fruits obtained in Chester County, PA, USA. Both mushrooms had similar total As concentrations of 0.37 μg g−1. Color scale corresponds to fluorescence intensity for each element. Numbered boxes in A and B correspond to locations of higher resolution images shown in Figure 3.
mushrooms showed the largest range in values for As, Asi, and Pb, but not for Cd (Figure 1; Table S2−5). Total As concentration ranged 0.1−1 μg g−1 among all samples and was significantly higher in Cremini compared to Shiitake and Royal Trumpet (Figure 1A). Of the samples analyzed for As speciation (i.e., those higher than 0.4 μg g−1 total As), Asi was the major component and was 25−94% of the total As for
ANOVA assumptions. All statistical tests were conducted using with SPSS v.23 software.
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RESULTS Market Survey. Concentrations of As, Pb, and Cd were less than 1 μg g−1 (d.w.) in all collected mushrooms, but varied within and among mushroom types (Figure 1). White button D
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Figure 3. High resolution X-ray fluorescence images in two locations depicted by numbered boxes in Figure 2 in a Shiitake mushroom fruit showing localization of Ca and As in 30-μm-thin sections. Color scale corresponds to fluorescence intensity for each element.
most samples while DMA and AsB ranged up to 28 and 20%, respectively, of the total As (Table 1). Portobello had significantly less inorganic As than Cremini (Figure 1B). Pb concentrations were less than 0.16 μg g−1 for most samples with the exception of two samples of white button collected from the farm that was utilized in the substrate-to-mushroom sampling survey, which had Pb levels higher than 0.75 μg g−1; even when removing those outliers, no significant differences in Pb levels were found among mushroom types (Figure 1C). Cd concentrations were less than 0.3 μg g−1 in most samples, yet levels in White Button, Cremini, and Portobello were significantly lower than in Oyster mushrooms (Figure 1D). As Localization. One Shiitake and one Cremini that had similar total As concentrations of 0.431 and 0.444 μg g−1, respectively, and were collected from the same farm show differences in As localization between these mushroom types (Figure 2). Using Ca as an orientation guide (Figure 2A, C), it is evident that As is localized to the hymenophore in Shiitake (Figure 2B) and is distributed more uniformly throughout Cremini fruit (Figure 2D). Higher resolution images of Shiitake Ca and As localization depicted in yellow boxes 1−4 in Figure 2A, B reveal a strong correlation between Ca and As in the veil remnants, but exclusion of As in the gills and cap margin (Figure 3). Substrate-to-Mushroom Transfer and Bioaccumulation. Starting materials of mushroom substrate collected from one farm in Chester County, PA, show differences in the concentrations of As, Cd, and Pb that range at least an order of magnitude (Table 2). The highest concentration of both As and Pb were found in the gypsum (0.70 and 14 μg g−1, respectively) which was sourced from recycled dry-wall material. Pb concentrations were also elevated in peat moss (5.0 μg g−1), chicken manure (1.1 μg g−1), and spent substrate that is blended into the new substrate compost (1.8 μg g−1). After gypsum, the highest levels of As were found in chicken manure
Table 2. Bioaccumulation Factors and Dry-Weight Concentration of As, Cd, and Pb in Mushroom Substrate Compost Starting Materials, Compost at Various Stages of Development, and Final Products Collected from One Mushroom Facility in Chester County, PA, USA μg g−1 mushroom substrate and final products Compost Starting Materials gypsum (recycled dry wall material) chicken manure (feathers removed) chicken feathers (separared from manure) corn cob peat moss cocoa hulls spent substrate with hyphae (blended into compost) horse manure cottonseed hulls cottonseed meal timothy and orchard grass hay mix wheat straw Compost Development prewet midcompost nearly finished compost final compost Substrate to Fruit substrate White Button hyphae White Button fruit (large fruit) White Button fruit (small fruit) White Button bioaccumulation factor
As
Cd
Pb
0.70 0.29 0.26 0.25 0.22 0.20 0.18 0.14 0.08 0.04 0.03 0.02
0.10 0.29 0.36 0.05 0.12 0.54 0.07 0.08 0.16 0.15 0.21 0.14
13.8 1.08 0.50 0.34 4.97 0.57 1.81 0.88 0.27 0.06 0.15 0.12
0.22 0.20 0.20 0.28
0.13 0.11 0.10 0.12
2.93 3.48 4.45 2.58
0.20 0.14 0.17 0.17 0.86
0.09 0.12 0.15 0.14 1.53
4.93 0.45 0.78 0.92 0.19
(0.29 μg g−1), feathers (0.26 μg g−1), and corn cob (0.25 μg g−1). Cd levels were highest in cocoa hulls (0.54 μg g−1) and were also elevated in chicken feathers (0.36) and manure (0.29 E
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Figure 4. X-ray fluorescence images of As (A), Ca (B), Fe (C), and P (D) in White Button mushroom substrate and hyphae. Total As concentrations in substrate, hyphae, and fruit were 0.20, 0.14, and 0.17 μg g−1, respectively, and are reported in Table 1. In panel B, s refers to substrate and h refers to hyphae.
μg g−1). The distribution of As in White Button mushroom substrate shows the presence of As in small hyphae (see arrow in Figure 4B) and absence in larger hyphae >0.25 mm in diameter (Figure 4). The levels of As, Cd, and Pb in developing compost, substrate, and fruit led to different bioaccumulation factors in White Button mushrooms (Table 2). While Cd concentrations in starting materials and substrate are lower than Pb and As concentrations, the proportional accumulation into the mushroom fruit was greater and resulted in a bioaccumulation factor of 1.53 for Cd and only 0.86 and 0.19 for As and Pb, respectively. Note that the concentrations of Pb used to calculate the Pb bioaccumulation factor were the highest found in our market survey by nearly an order of magnitude.
surveys;31,49 however, As, Pb, and Cd in our survey are lower than those reported in a recent Chinese market survey30 in which median metal(loid) concentrations in L. edodes (Shiitake), P. ostreatus (Oyster), and A. bisporus (White Button, Cremini, Portobello) were up to 2-fold higher for As and Cd, and up to 40-fold higher for Pb than we observed (Tables S1, S3, S4). While the concentrations of As, Pb, and Cd in cultivated U.S. mushrooms in our study were low and ≤1 μg g−1, they were different from those observed in the TDS from 1994−2011 (Figure S3 and Tables S1−S4). The TDS reported As, Pb, and Cd concentrations in raw mushrooms from nondetectable up to 0.2, 0.04, and 0.02 μg g−1, respectively, on a fresh-weight basis. Converting to a dry-weight basis by accounting for an average of 90% water,20 this equates to 2.0, 0.4, and 0.2 μg g−1 for As, Pb, and Cd, respectively, but Pb was only detected in three samples in the TDS. In contrast, we detected these elements in all analyzed samples and while we report similar concentrations for total As, we found up to 2-fold higher concentrations for Pb and Cd. Previous work20 has revealed a wide range of metal(loid) concentrations among different mushroom species and within one species due to differences in the age of the mycelium, wave of mushroom harvest, and metal(loid) content of the substrate, which may account for the differences we observed between our study and the TDS. Alternatively, metal(loid)s may have been concentrated in the drying process as water was removed prior to microwave digestion in our study, allowing for increased signal intensity, whereas fresh mushrooms analyzed in the TDS would have had a dilution effect resulting in nondetects for Pb. Nevertheless, U.S. mushrooms contain relatively low levels of As, Pb, and Cd. Despite small sample sizes per mushroom types, some significant difference were observed in metal(loid) content between mushroom types, whereas no difference was observed between geographic sampling locations in PA and CA. Cremini
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DISCUSSION Trends in Cd, Pb, and As in Mushrooms. The concentrations of As, Pb, and Cd in cultivated U.S. mushrooms reported here are among the lowest that have been reported to date. The concentrations of As, Pb, and Cd we observed are much lower than those that have been reported for wild mushrooms near contaminated sites19,45 and are similar to or lower than those reported for wild mushrooms growing in noncontaminated sites,19,46,47 which typically range 0.5−50 and 0.5−10 μg g−1 d.w for Cd and Pb, respectively, while As concentrations are typically less than 5 μg g−1 d.w but can be as high as 300 μg g−1 d.w. in some species.20 In contrast, cultivated mushrooms having had less time of exposure to metal(loid)s in substrate have much lower concentrations of metal(loid)s in fruit compared to wild mushrooms.19,20,30,31,42,48 We observed As, Pb, and Cd concentrations similar to or lower than those reported for some cultivated mushrooms.30−32,48,49 The concentration of total As we observed are within the range reported from a recent Canadian market survey32 and are slightly lower than those reported in recent Spanish market F
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Cremini or Shiitake were evaluated in that study, our Shiitake data seem to coincide with their report. However, upon higher resolution imaging analysis (Figure 3), arsenic appears to be excluded from the gills (spore-forming) and instead localized to the veil remnants on the underside of the cap in Shiitake. In the Arvay et al.21 study, the hymenophore was physically removed from the rest of the fruiting body and each portion separately analyzed, whereas we utilized X-ray fluorescence imaging on whole-mushroom thin sections to reveal As localization, which gives more detailed information. Arsenic was localized similarly to Cu and Zn in both Shiitake and Cremini (Figure S4.). This new data suggests that Shiitake mushrooms are capable of concentrating metal(loids) away from their reproductive regions, whereas Cremini mushrooms distribute metals more uniformly throughout the fruit. Our data suggest that removing the veil remnants from the underside of the Shiitake cap could decrease potential ingestion of As, whereas doing this would have no impact on As ingestion from Cremini. Further investigation into localization of metal(loid)s among different mushroom fruits is warranted. We observed lower proportions of AsB (nondetectable to 14% of total As) and higher proportions of Asi (5−94% of total As) than have been reported previously for wild and cultivated mushrooms, supporting previous work that As speciation differs for the same mushroom species collected from different areas and/or grown on different substrates.51,29 In contrast to our study, Nearing et al.32 reported mostly AsB and lesser amounts of DMA and Asi in A. bisporus collected in a Canadian market survey, which may have been due to differences in the Asi content of mushroom growth substrate between the two studies. U.S. mushrooms from Chester County, PA, where most U.S. mushrooms are grown, are typically cultivated on substrate containing 7−10% poultry litter, which may contain elevated levels of Asi after composting if poultry were fed Asbearing additives.33 While the use of organic arsenicals are being phased out in U.S. poultry, they were still in use at the time of our sample collection and may explain the elevated Asi observed in A. bisporus cultivated in the U.S. A follow-up study after the full phasing out of organic arsenicals in poultry is warranted. Mushroom fruits vary in As species distributions due to substrate differences as well as among mushroom species and cultivars. In our study, the highest Asi levels were observed in A. bisporus and within that species, Cremini had significantly higher Asi than Portobello, which may be the result of more As transformation from inorganic to organic species in substrate as duration of growth increases. The transformation of Asi to AsB appears to be controlled by microbial transformations in substrate rather than in vivo transformation.32,52 A. bisporus grown on a substrate amended with 1000 μg g−1 As(V) contained mostly (98%) Asi within the edible portion and had much higher As(III) compared to As(V) (despite growing on substrate amended with As(V)); however, mushrooms grown on a nonamended (total As = 3.8 μg g−1) substrate had higher amounts of the nontoxic arsenobetaine and lesser amounts of DMA, MMA, and the Asi compounds.28 In another study, mushrooms cultivated on substrate with ca. 200 μg g−1 either from mine-waste or an arsenate solution contained mostly arsenobetaine.42 The discrepancy between these studies suggests arsenobetaine formation in substrate may depend on the initial composition (As concentration and speciation) of the substrate, since arsenobetaine was not detected in mushrooms growing on high-As substrate. While As speciation in our study
mushrooms clustered on the higher range for total As and Asi and were significantly higher in total As than Shiitake and Asi than Portobello. Portobello and Cremini are the same species, but the former is allowed to grow for a longer period of time, which may allow longer time for in situ transformation of inorganic As to less toxic organic forms in addition to a potential dilution effect due to biomass differences. Shiitake is cultivated on logs, which contain low levels of As, whereas Cremini is cultivated in compost substrate, which may be a source of As depending on the As content in compost starting materials. Importance of Substrate. Growing practices and particularly substrate metal(loid) content are important for determining total metal(loid) concentration in mushroom fruit.30,48,50 In our study, both the lowest and highest concentrations of As, Asi, and Pb were observed in the common White Button mushroom collected from different farms with different mushroom growth substrate. In addition, the two samples with the highest Pb levels were obtained from the farm from which we also obtained all compost starting materials for these White Button mushrooms; these results revealed surprisingly elevated Pb levels in the gypsum sourced from recycled drywall board (Table 2). We suggest that Pbbased paint may have been used on the drywall board prior to collection that became available for uptake into the fungal mycelium upon compost preparation. The Pb BAF of 0.19 at this farm was much higher than previously reported for other environments, which is typically
