Javier Peinador Asensio

In this study, 48 different wild and cultivated mushrooms were purchased in local markets in Madrid and provided by trusted specialists from the Mycological Society of Madrid. Cadmium, mercury, and lead were determined by inductively coupled plasma–mass spectrometry (ICP-MS) in samples previously digested in a microwave oven. Methylmercury (MeHg) was determined by liquid chromatography separation (HPLC) combined with ICP-MS after microwave-assisted extraction. Regarding the cadmium and lead contents, all the samples complied with the maximum permitted limits. For mercury, a single sample exceeded the maximum value established in Regulation (EU) 2018/73 for wild mushrooms. In the case of methylmercury, the highest amount represents 13% of the total mercury in one sample. In the rest of the cases, the concentration found was negligible.

Historically, fungi have been classified as inferior plants in the division 

 because of their similarities to other members of that group (no roots, no leaves). However, later studies demonstrated unique features that led to the formation of the new kingdom, the Kingdom Myceteae, which includes microscopic and macroscopic species of fungi. Mushrooms are described as large multicellular fungi with stems and caps. According to Chang and Milles, mushrooms can be simply defined as “macro fungi with a distinctive fruiting body and large enough to be seen with the naked eye and to be picked by hand” (1).

Edible mushrooms have always been a popular food in diets around the world. Their texture and flavor make them highly appreciated in the kitchen. Consuming mushrooms brings multiple benefits because they are rich in protein, dietary fiber, vitamins and minerals, and low in fat. In addition, they contain antioxidant compounds such as polysaccharides, terpenoids, and polyphenols, all of which have a positive impact on health (2,3)

However, the scientific literature shows that mushrooms can accumulate heavy metals present in the soil. The fungal mycelium plays an essential role in this, which spreads over the available area and incorporates metal ions into its cytosol under suitable conditions. Mushrooms are able to accumulate higher concentrations than plants because, unlike their roots, their mycelium develops mainly horizontally, normally occupying the most superficial part (5–10 cm) (4). The mycelium contains the highest concentration of heavy metals (5). Although this characteristic makes them extremely useful as bioindicators of contaminated areas (6), edible mushrooms may pose a risk to consumers if the heavy metal content exceeds reference levels.

The main factors that influence the bioaccumulation of heavy metals in these organisms are environmental factors, which are the concentrations of metals in the soil, the pH, the amount of organic matter and contamination by atmospheric deposition, and intrinsic fungal factors of the species, such as its structure, biochemical composition, decomposition activity, and development of mycelium and fruit bodies (7,8).

Among the heavy metals, it is worth mentioning mercury, cadmium, and lead, which are considered toxic elements for human health by the European Food Safety Authority (EFSA).

Mercury is a toxic heavy metal that can bioaccumulate not only in marine organisms (mainly swordfish and sharks), but also in fungi. There has always been significant concern about its exposures, intakes, and toxicity. In the environment, mercury can be found in three different chemical forms: elemental/metallic mercury (Hg

), and organic mercury (organometallic compounds where one or two alkyl/aryl substituents are attached to the mercury atom). These forms present different bioavailabilities and toxicity, with methylmercury being the most dangerous in the food chain (9).

To perform an accurate risk assessment, it is necessary to collect as much information as possible on the presence of these forms in food because exposure to methylmercury can cause harm by interacting with the sulfhydryl (-SH) groups distributed throughout the body, causing the well- known Minamata disease (6).

The central nervous system is the most important target of methylmercury, which can cause different symptoms, including sensory disturbances in the hands and feet, ataxia, and visual impairment. In addition, organic mercury can reduce the activity of natural killer (NK) cells and generate free radicals. Acute inorganic mercury poisoning affects the gastrointestinal tract and kidneys, causing severe diarrhea, vomiting, and renal tubular necrosis with anuria (6).

In the 2012 EFSA opinion, the tolerable weekly intake (TLWI) for methylmercury was updated to 1.3 μg/kg bw and 4.0 μg/kg bw for inorganic mercury, which is the maximum amount that a person can ingest weekly throughout their lives without adverse effects (9).

European legislation establishes maximum Hg contents in mushrooms. Commission Regulation (EU) 2018/73 distinguishes between cultivated and wild mushrooms, establishing maximum limits of 0.050 mg/kg and 0.50 mg/kg of Hg, respectively. It also establishes a maximum limit of 0.90 mg/kg in 

Cadmium is also a heavy metal that poses serious risks to human health. Until recently, it has not been possible to demonstrate that cadmium has any physiological function, so its main interest lies in its high toxicity in humans, especially in its inorganic forms (11).

Various studies have shown that exposure to cadmium through diet can cause harmful effects on health, mainly in the kidney, spleen, and liver where it accumulates (12). In addition, it is currently classified by the International Agency for Research on Cancer (IARC) as carcinogenic to humans (Group 1), as an association between its intake and the risk of postmenopausal endometrial cancer has been demonstrated. As a result of its ability to induce oxidative stress, it can also affect male fertility by decreasing sperm quality (13).

Prolonged ingestion of cadmium also poses a serious health risk, by causing Itai-Itai disease, the best-known case of which was the mass poisoning that took place in Japan in the last century. This disease is characterized by creating bone damage due to osteoporosis and osteomalacia, in addition to severe kidney damage (14,15). Derived from a comprehensive toxicological assessment, EFSA has established a tolerable weekly intake (TSI) for cadmium of 2.5 μg/kg body weight (16).

In 2023, the maximum cadmium content in mushrooms has been updated by Regulation (EC) No 2023/915, establishing the maximum limit at 0.050 mg/kg for cultivated mushrooms, at 0.15 mg/kg for the mushrooms 

 (oyster mushroom), and 0.50 mg/kg in wild mushrooms (17).

Finally, lead is another heavy metal to consider. Lead contamination arises primarily from the environment, through atmospheric lead through deposition on agricultural crops, from water, or from food processing, handling, and packaging. Although lead exists in both organic and inorganic forms, only inorganic lead has been detected in food (18). When lead is ingested, it is distributed to certain organs such as the brain, kidneys, liver, and bones, causing tissue damage. After several weeks, most of it moves to the bones and teeth, where it can be stored for decades (19,20).

Accumulated lead can return to the blood, reaching vital organs, resulting in episodes of broken bones in the elderly. During pregnancy, accumulation of lead can lead to possible effects on the fetus and lactation (20).

Lead poisoning produces adverse effects at the central nervous system level, which can affect brain development and cause behavioral changes such as reduced attention span and increased antisocial behavior. In addition, it can lead to anemia, hypertension, renal failure, immunotoxicity, and toxicity to reproductive organs (19).

Due to the lack of sufficient scientific information, in 2010 the existing toxicological safety threshold for lead was withdrawn, and a new one could not be determined. Therefore, there is currently no recommended tolerable intake for lead (21).

As of this year, the maximum lead content in mushrooms has been updated by Regulation (EC) No. 2023/915, establishing the maximum limit at 0.30 mg/kg for 

 (shiitake mushroom) mushrooms, and 0.80 mg/kg in wild mushrooms (17).

The main objective for this study is to provide information on the content of cadmium, lead, mercury, and methylmercury in both cultivated and wild mushrooms and to compare the results obtained with the limits established in current legislation. For this, a total of 48 mushrooms were obtained and analyzed.

For the analysis of cadmium, lead, and total mercury, the inductively coupled plasma mass spectrometer (ICP-MS) (Agilent Technologies model 7900) was used. In addition, an integrated sample introduction system (ISIS 3) and an automatic sampler (SPS 4) were used in the determination together with the ICP-MS. The operating parameters of the ICP-MS analysis are shown in Table I.

Methyl-mercury determinations were performed using a liquid chromato- graph (HPLC) (Agilent Technologies 1200 Infinity), consisting of a model 1260 Quaternary Pump VL, a model 1260 ALS injector, and a model 1260 TCC column compartment. This HPLC system is coupled to the ICP-MS described above. The characteristics are detailed in Table II.

For digestions and extractions, a DigiPREP Jr digestion block from SCP Science with 24 positions was used. The volumetric material used for all the solutions were DigiTubes, polypropylene tubes single-use, except in the case of MeHg analysis, which opted for centrifuge tubes, also single-use, VWR Metal Free, 15 mL, and volumetric flasks of 1 L and 200 mL capacity. To take the cor

responding aliquots, Eppendorf brand micropipettes were used.

For centrifugation of the samples, a Lisa refrigerated centrifuge of AFI groups was used.

For the preparation of the reagents, dilutions, washing, and rinsing solution of the work material, deionized water (H

O) of Class Type I (according to the UNE-EN-ISO 3696 Standard) was used, purified with the Milli-Merck’s Q Comprehensive. Nitric acid (HNO

, 65%) from Scharlau was distilled in the laboratory using Savillex equipment, model DST-1000. Suprapur hydrochloric acid for trace analysis (HCl, 37%) supplied by Scharlau and hydrogen peroxide (H

Regarding the ICP-MS, the tuning solution containing 1 μg/l Ce, Co, Li, Mg, Tl, and Y was used. Finally, argon and helium (99.999%) for plasma generation and interference removal.

Standards of 1000 mg/L of Cd (Scharlau), Pb (VHG-Labs), Hg (Scharlau), and MeHgCl (VHG-Labs) were also used. For the determination of cadmium, lead, and total mercury, the intermediate solutions and the calibration curve were prepared with a 2% HNO

As an internal standard, a 0.4 mg/L iridium and rhodium solution (from a 1000 mg/L Scharlau standard) was prepared in a 2% HNO

, 1% HCl, and 5% acetic acid solution for the determination of cadmium, lead, and total mercury. To stabilize the signals, 0.4 mg/L Au from SCP Science was added. No internal standard was used for the methylmercury speciation method.

The composition of the mobile phase was 1 g/L L-cysteine/20 mM CH

/20 mL MeOH in deionized water, with a pH of 6.9, using L-cysteine from Acros Organics (minimum purity 98%), ammonium acetate (CH

) from PanReac AppliChem (96% purity), and methanol (MeOH) from Scharlau (GC residue analysis grade, 

In the case of the mercury speciation analysis, the solution used for the extraction was 5 g/L L-cysteine/0.5% HCl, and the solution used for the preparation of all the intermediate solutions and the calibration curve was the mobile phase.

The samples were homogenized by crushing with a mincer, until a representative sample of the product to be tested was obtained. Two different methods were carried out in this study. Both represented in Figure 1.

FIGURE 1: Schematic diagrams of the different methods (total heavy metals and methylmercury) used for this study.

Regarding the total analysis of mercury, cadmium, and lead, first 0.5 ± 0.05 g of sample were weighed in a DigiTube, then 3 mL HNO

 were added, and the samples were digested in the DigiPrep afterwards. Once the digestion was complete and the samples had reached room temperature, they were made up to volume in 50 mL with deionized water. The sample collection were analyzed by ICP-MS, samples with high analyte contents that were diluted previously.

For methylmercury speciation, 0.2 ± 0.02 g of sample was weighed into a DigiTube, 10 mL of extraction solution (1 g L-cysteine; 5% HCl) was added, and extraction was performed on the DigiPrep for 30 min at 60 

C (Figure 2). Once the process was finished, the samples were allowed to cool to room temperature, transferred to 15 mL tubes, and centrifuged for 10 min at ≥ 4000 rpm and 4 

C. Subsequently, the supernatant was filtered using 0.45 μm pore size filters and placed in 1-mL polypropylene vials to be injected into the HPLC system.

FIGURE 2: Temperature profile for methylmercury speciation. For sample preparation, 0.2 ± 0.02 g of the sample was weighed into a sample tube, 10 mL of extraction solution (1g /L-cysteine; 5% HCl) was added, and extraction was performed for 30 min at 60 °C.

Figure 3 shows a chromatogram where you can see the separation of the peaks obtained, where the first corresponds to Hg, with a retention time of 2.384 min, and the second to MeHg, with a retention time of 3.868 min.

FIGURE 3: The chromatogram illustrates the separation of the peaks obtained, where the first corresponds to Hg, with a retention time of 2.384 min, and the second to MeHg, with a retention time of 3.868 min.

For the calibration curves used in the quantification of the analytes, the 

-axis represents the value of the ratio, calculated as the number of counts of the analyte divided by the number of counts of the internal standard, whereas the 

-axis represents the concentration in μg/L. In the case of MeHg, the 

-axis represents the number of counts per area of the chromatographic peak.

Figures 4, 5, 6, and 7 show the calibration curves used in the quantification of the samples.

FIGURE 4: Calibration curve (with statistics) used in the quantification of cadmium (Cd).

FIGURE 5: Calibration curve (with statistics) used in the quantification of lead (Pb).

FIGURE 6: Calibration curve with statistics used in the quantification of mercury (Hg).

FIGURE 7: Calibration curve with statistics used in the quantification of methylmercury (MeHg).

The test method used for the determination of heavy metals meets the criteria of Regulation (EC) 333/2007 of the Commission of March 28, 2007, which establishes the sampling and analysis methods for the control of levels trace elements and process contaminants from food products and their modifications. The validation was carried out according to the requirements of the UNE/EN-ISO 17025 Standard for Testing Laboratories, and it is accredited by the National Accreditation Entity (ENAC). For MeHg determination, the test method is in the accreditation process.

A total of 48 wild and cultivated mushrooms were acquired and analyzed. All of them were purchased in local markets in Madrid and provided by trusted specialists in the manual collection of mushrooms from the Mycological Society of Madrid.

The results obtained in the study of cadmium, lead, and total mercury of the 48 samples analyzed are shown in Table III.

Regarding the cadmium and lead contents, all the samples comply with the maximum permitted limits, and do not seem to pose a risk to the population.

Regarding the total mercury content, it should be noted that a single sample (

 with a content of 0.97 mg/kg) exceeded the maximum value established in Regulation (EU) 2018/73, set at 0.50 mg/kg for wild mushrooms. No sample of 

 reached the maximum allowed limit of 0.90 mg/kg for said species. In the case of samples of cultivated mushrooms, it is observed that their Hg content is less than 0.050 mg/kg established by the aforementioned regulation.

According to the data obtained, it can be observed that cultivated mushrooms have significantly lower concentrations of metals than wild mushrooms.

The results of the methylmercury determination are shown in Table IV. In this case, only the samples with a significant total mercury content were analyzed. Speciation was performed in the 10 samples that exceeded 0.075 mg/kg of total Hg, all of them wild. The highest amount of MeHg found was 0.12 mg/kg in a sample of 

, which represents 13% of the total mercury in the sample.

It can be determined that the test method used for the analysis of Cd, Hg, and Pb by acid digestion and determination by inductively coupled plasma–mass spectrometry is considered a valid option to carry out a study, such as the one presented here, obtaining results in accordance with the requirements of ISO 17025.

In the same way, the coupling of a liquid chromatograph to an ICP-MS is a recommended method to perform the chromatographic separation of the different mercury species, and to be able to achieve the methylmercury analysis. This method is in the accreditation process.

After determining the content of cadmium, lead, and total mercury and the speciation of methylmercury in the samples analyzed, it can be concluded that the amounts found in the case of cultivated mushrooms are lower than the limits established in European legislation, whereas in the case of wild mushrooms, a result was obtained above the established limits, which may mean a risk to the population.

The values obtained by performing the mercury speciation reveal that the mercury content in the mushrooms is almost entirely inorganic mercury and the amounts of methyl mercury found are practically negligible, becoming, in the worst case, less than 13% of total Hg.

We would like to thank the Mycological Society of Madrid for its invaluable role in providing us with samples of wild mushrooms in order to develop the study of their metal content.

Mushroom: Cultivation, Nutritional Value, Medicinal Effect, and Environmental Impact

(2) Gupta, S.; Summuna, B.; Gupta, M.; Annepu, S. K. 

; Springer International Publishing AG, 2018; pp 1815–1847.

(3) Heleno, S. A.; Barros, L.; Martins, A.; et al. Chemical Composition, Antioxidant Activity, and Bioaccessibility Studies in Phenolic Extracts of Two Hericium Wild Edible Species. 

(5) Berthelsen, B. O.; Steinnes, E. Accumulation Patterns of Heavy Metals in Soil Profiles as Affected by Forest Clear-Cutting. 

(6) Bernhoft, R. A. Mercury Toxicity and Treatment: A Review of the Literature. 

(7) García, M. A.; Alonso, J.; Fernández, M. I.; Melga, M. J. Lead Content in Edible Wild Mushrooms in Northwest Spain as Indicator of Environmental Contamination. 

(8) Lalotra, P.; Gupta, D.; Yangdol, R.; Sharma, Y. P.; Gupta, S. K. Bioaccumulation of Heavy Metals in the Sporocarps of Some Wild Mushrooms. 

https://www.efsa.europa.eu/en/efsajournal/pub/2985

(10) European Commission. Commission Regulation (EU) 2018/73 of 16 January 2018 Amending Annexes II and III to Regulation (EC) No 396/2005 of the European Parliament and of the Council as Regards Maximum Residue Levels for Mercury Compounds in or on Certain Products. 

https://eur-lex.europa.eu/collection/eu-law/consleg.html

(11) Godt, J.; Scheidig, F.; Grosse-Siestrup, C.; et al. The Toxicity of Cadmium and Resulting Hazards for Human Health. 

(12) Kalač, P.; Svoboda, L.; Havlíčková, B. Contents of Cadmium and Mercury in Edible Mushrooms. 

(13) Ciobanu, C.; Şlencu, B. G.; Cuciureanu, R. Estimation of Dietary Intake of Cadmium and Lead through Food Consumption. 

(14) Satarug, S. Dietary Cadmium Intake and Its Effects on Kidneys. 

(15) Inaba, T.; Kobayashi, E.; Suwazono, Y.; et al. Estimation of Cumulative Cadmium Intake Causing Itai–Itai Disease. 

(16) Cadmium in Food—Scientific Opinion of the Panel on Contaminants in the Food Chain. 

(17) European Commission. Commission Regulation (EU) 2023/915 of 25 April 2023 on Maximum Levels for Certain Contaminants in Food and Repealing Regulation (EC) No. 1881/2006. 

Evaluation of Certain Food Additives and Contaminants: Seventy-Third Report of the Joint FAO/WHO Expert Committee on Food Additives

; WHO Technical Report Series; 960; Geneva, Switzerland, 2011.

(19) World Health Organization. Lead Poisoning and Health. 2022. 

https://www.who.int/news-room/fact-sheets/detail/lead-poisoning-and-health

(20) Agency for Toxic Substances and Disease. Public Health Statement Lead CAS# 7439-92-1. 

https://wwwn.cdc.gov/TSP/PHS/PHS. aspx?phsid=92&toxid=22, 2007

(21) Scientific Opinion on Lead in Food. 

 is with the Public Health Laboratory, in Madrid, Spain. Direct correspondence to: 

Articles

The bioaccumulation of heavy metals can lead to disastrous effects on the environment and human health. Here, ICP-MS was applied to study 48 different mushrooms collected at local markets in Madrid, Spain, showcasing its effectiveness at detecting heavy metals in edible fungi.

Study of Heavy Metals and Methyl-Mercury in Fungi in Markets of Madrid

In this study, 33 different dried seaweed products were purchased in local markets in Madrid and analyzed. Arsenic, cadmium, mercury, nickel, lead and iodine were determined by inductively coupled plasma–mass spectrometry (ICP-MS) in samples previously digested in a microwave oven. In addition, inorganic arsenic (iAs) was determined by high performance liquid chromatography (HPLC) combined with microwave-assisted extraction followed by ICP-MS. Results indicate that the levels of Cd, Ni, and Pb were relevant in some of the samples, and may pose a risk to consumers. The concentrations of Hg were overall the lowest found in the samples. Hijiki seaweed (

) contained a considerable iAs fraction, whereas all the other seaweed species contained minimal fractions. Lastly, iodine content in most of the seaweeds surpassed the tolerable daily intakes recommended by the European Food Safety Authority (EFSA), which may pose a risk to consumers.

Seaweed has been a part of Asian diets since ancient times, but only more recently has it become a popular ingredient in Western diets because of its potential as an alternative and healthy source of food. Other applications of seaweed are also being explored, such as in food additives, feeds, fertilizers, biofuels, cosmetics, and medicines (1).

Current research on the health benefits of seaweed reveals that it contains various important vitamins (A, B1, B12, C, D, and E), minerals (potassium, sodium, phosphorus, iodine), and compounds with remarkable antioxidant capacities (2–5). Seaweed also has a low-calorie content (between 1% and 5% dry weight) (5).

However, some studies highlight that there are adverse effects to consuming large quantities of seaweed because it contains an excessive amount of heavy metals, arsenic, and iodine. As a result, there are possible risks associated with consuming too much seaweed (6).

Heavy metals not only have no beneficial function in the human body, but they are also considered toxic for humans even at low concentrations. Exposure to heavy metals usually leads to negative gastrointestinal, neurotoxic, and carcinogenic effects (7).

It is widely known that marine organisms, especially seaweed, accumulate relevant amounts of arsenic. The chemical similarities between arsenate (H

-) is the reason why both are absorbed equally through membrane transport systems. Thus, inorganic arsenic (As

) is introduced in these organisms and metabolized to organic arsenic species, such as arsenobetaine, monomethylarsonic acid, dimethylarsinic acid, arsenosugars, and arsenolipids (8,9). It is reported that inorganic forms of arsenic are more dangerous and toxic than organic forms of arsenic (10). For that reason, a suitable speciation analysis must be carried out to determine the levels of inorganic arsenic in seaweed.

Iodine is a diet-essential mineral because of its importance in the thyroid gland. It serves important biological and physiological functions in the human body, especially during early growth and organ development (11). Although iodine is necessary in the diet, either an excess or a deficiency in iodine may cause disorders related to the thyroid functions. The consequences of these pathologies can be very serious in children and pregnant women (12,13).

Regulation (EU) 2017/2470 established and updated the list known as the “Novel Food Catalogue” that contains all European Union’s authorized novel foods. This list included 22 different types of edible seaweed by the end of 2020, but does not specify the maximum limits of heavy metals, iodine, and arsenic (14).

Food supplements that are seaweed-based are regulated by Commission Regulation (EC) N

 629/2008, which indicates the maximum amount of cadmium that food products can contain (15). However, there is currently no specific legislation about maximum limits related to seaweed as food yet. Therefore, the Europe Union published 

 on the monitoring of different elements, such as arsenic, cadmium, lead, mercury, and iodine, in edible seaweed. 

 also mentions the importance of quantifying different species of arsenic (As

, and total arsenic) and, if possible, other relevant arsenic species. The aim for this recommendation is to gather results from different studies, which will allow us to assess the risks associated with the consumption of seaweed, and consequently set maximum levels in the future (16).

Inductively coupled plasma–mass spectrometry (ICP-MS) is a fitting technique to quantify several elements in foodstuffs at low concentrations simultaneously. However, it is important to notice several issues in using ICP-MS when analyzing inorganic arsenic and iodine.

Total arsenic and heavy metals analysis are commonly the same; the process for both first undergoes an acid digestion followed by quantification using an elemental analysis instrument. Nevertheless, inorganic arsenic analysis has significant differences that need to be considered. A regular acid digestion is not suitable because organic arsenic species would deteriorate into As

. Thus, it is mandatory to utilize a previous extraction followed by a separation technique. High performance liquid chromatography (HPLC) is the most common methodology for arsenic speciation because it allows for the separation of individual arsenic species so they can be quantified accurately afterwards (17).

The literature highlights the problematic sample preparation of iodine because of memory effects and its complex redox chemistry. Equation 1 reveals that at low pH (typical digestion mixture where H

) is oxidized to volatile molecular iodine (I

 > 0 V), thus a regular acid digestion would lead to iodine loss and an incorrect approach to sample pretreatment, which why methods involving alkaline digestion are widely used (18,19). An alternative method being proposed in this study is performing a suitable acid digestion followed by a dilution using an alkaline solution, which is an unique and simultaneous sample pretreatment. Performing a suitable acid digestion followed by a dilution using an alkaline solution saves time and resources while taking advantage of ICP-MS multielemental analysis (20).

The cadmium, mercury, lead, nickel, total arsenic, and iodine totals were determined using an ICP-MS instrument (Agilent Technologies model 7900). An Integrated Sample Introduction System (ISIS 3) and a SPS 4 autosampler, both from Agilent Technologies, were used alongside the ICP-MS instrument, in determining the quantities of the elements mentioned above. The ICP-MS analysis operating parameters are shown in Table I.

Inorganic arsenic was determined using a HPLC instrument (Agilent Technologies model 1200 Infinity) equipped with a model 1260 Quat Pump VL quaternary pump; a model 1260 ALS injector; and a model 1260 column compartment. The HPLC system was used in conjunction with the ICP-MS instrument mentioned earlier. For the inorganic arsenic determination, the operating parameters are listed in Table I.

Samples were treated in a Milestone model Ethos One microwave oven with a capacity of 10 polytetrafluoroethylene (PTFE) vessels at 100 mL each. One of the PTFE vessels is always used for a blank process.

The volumetric materials used were calibrated with polypropylene single-use tubes (DigiTubes from SCP Science), 15 mL single-use centrifuge tubes (Superclear Labcon), and a 1 L volumetric flask to prepare the mobile phases.

To centrifuge the samples, a refrigerated Lisa centrifuge from AFI groups was used.

All solutions, cleaning procedures, digestion, and extraction methods were prepared with deionized water (H

O) class type I (according to UNE-EN-ISO 3696), and purified using a Milli-Q system from Merck. Nitric acid (HNO

 65%) pro analysis (p.a.) quality was used, supplied by Fluka and previously distilled by a Savillex distiller model DST-1000. Hydrocloric acid (HCl 37%) was ultratrace ppb-trace analysis grade from Scharlau, and hydrogen peroxide (H

, 30%) was suprapur grade and supplied by Merck.

Regarding the ICP-MS instrument, the tuning solution contained 10 μg/L cerium, cobalt, lithium, magnesium, thallium, and yttrium. The argon and helium used in plasma generation and interference elimination (KED mode) were both 99.999% accurate.

The alkaline solution used in the iodine determination was prepared with 40 g of NH

OH (Fluka p.a. quality), 10 g of 1-buthanol (Merck), and 1 g of ethylenediaminetetraacetic acid (EDTA) (Panreac pharma grade) mixed with 1 L of deionized water.

The extraction solution used in the inorganic arsenic determination was 0.2% HNO

. In arsenic and heavy metals analysis, standards of 1000 mg/L arsenic, cadmium, mercury, nickel, and lead (all supplied by LGC Standards) were used. Intermediate solutions and the calibration curve were prepared in 2% HNO

 and 1% HCl (v/v). Regarding the iodine determination, a 1000 mg/L iodine standard was provided by Scharlau. Suitable intermediate solution and calibration curve were prepared in a 1:2 diluted alkaline solution. For the inorganic arsenic determination, 100 mg/L As

 (LGC Standards) were utilized. Moreover, monomethylarsonic acid from High Purity Standards, dimethylarsinic acid from AccuStandard, and arsenobetaine from the National Metrology Institute of Japan (NMIJ) standards, were also used to check the chromatographic separation. The intermediate solutions and the calibration curve were prepared in 0.2% HNO

The internal standards used for each element in the ICP-MS analysis were germanium and rhodium from LGC Standards and iridium and tellurium from Sigma-Aldrich. A 0.4 mg/L mixture of these elements was prepared as the internal standard solution with 2% HNO

, 1% HCl, and 5% acetic acid. To stabilize the mercury signal, 0.4 mg/L of gold from SCP Science were also added.

The seaweed samples were purchased in several markets in Madrid, Spain. To obtain representative results, numerous species of seaweed were collected.

A total of 33 samples were analyzed in this study, including two dulse (

.) was also included in this study because of its use in foods and cuisine. Note that the conclusions related to species with fewer than three samples should be carefully considered.

All seaweed were homogenized first using a coffee grinder. The different methods are detailed in Figure 1.

FIGURE 1: A summary of the determination methods used in the study.

Determination of Iodine, Arsenic, and Heavy Metals

First, the homogenized samples were weighed into polytetrafluoroethylene (PTFE) vessels, and the digestion mixture of acids was added. Next, the samples were placed in the microwave oven. Once the digestion was complete and the samples cooled down to room temperature, they were transferred to DigiTubes and the tubes were filled with deionized water. An aliquot was taken and diluted with the alkaline solution (the ratio for the sample–alkaline solution was 1:2) for the parallel iodine analysis. These solutions were ready to be measured if no more dilutions were needed.

Inorganic arsenic is determined as the sum of arsenate (As

). The homogenized sample was weighed, mixed with the extraction solution, and introduced to the microwave oven. Validation proved that all As

 determined is equal to the complete inorganic fraction. After the microwave-assisted extraction, samples were centrifuged, diluted (if necessary), and filtered through a 0.45 μm polyvinylidene fluoride (PVDF) filters.

The determination methods of this work meet the criteria established in Commission Regulation (EC) N

Moreover, the laboratory is accredited according to ISO/IEC-17025 requirements. The studied heavy metals, total, and inorganic arsenic in foodstuffs are included in the schedule of accreditation.

Regarding the iodine determination, the analysis was not included in the schedule of accreditation then. Therefore, the laboratory participated in a proficiency test (FAPAS-Food Chemistry Proficiency Test 07380) to evaluate method accuracy and obtained a satisfactory result (

This work aims to provide information about the potential risks involved in seaweed consumption. For that purpose, the percentage contribution (%) by a daily portion of seaweed to a reference daily intake is estimated.

First, it is necessary to describe the typical portion of seaweed consumed in a day. Normally, labels on seaweed products give some instructions about how to cook them (an amount between 2 and 5 g is usually recommended). The literature also mentions a general daily intake of seaweed depending on the region. For example, the literature recommends estimating daily intake at 8 g/day in Asian cultures (4), 4–7 g/day in Japan (21), and <5.2 g/day in Western diets (22).

Second, a reference intake based on health impact is needed. European Food Security Authority (EFSA) scientific reports establish recommended intake values of the studied elements in foods considering their potential health risks. These values are often expressed in μg/kg bodyweight (b.w.) per day or per week, which means an average human bodyweight must be established to make the assessment.

Finally, once the results are obtained, the daily intake contribution for each element can be estimated using equation 2:

 is the highest result found of the corresponding element in seaweed (mg/kg), and the maximum value obtained allow to assess the worst-case scenario when discussing the results; 

 is the daily portion consumed of seaweed (kg/day); and TDI is the tolerable daily intake according to scientific reports published by EFSA (in mg/day). To estimate this contribution, a reasonable daily portion of 5 g and an average 60 kg (~132 lbs.) body weight are set.

The overall results are summarized in Table II. Concentrations, averages, and ranges obtained are shown as well as the number of samples quantified. The percentage of inorganic arsenic related to total arsenic is shown as well, and it is also displayed in the reference intakes from EFSA and its corresponding contributions (23–27). Moreover, individual results of each sample are shown in Figure 2.

FIGURE 2: Individual results of arsenic (As), iodine (I), and heavy metals, such as cadmium (Cd), lead (Pb), mercury (Hg), and nickel (Ni) in each seaweed species. 

-axis is the concentration results in mg/kg.

Heavy metals results show that mercury and nickel are not a threat in seaweed consumption because their contribution to the TDI was minimal (<1% and <6% for mercury and nickel, respectively). Regarding cadmium and lead, their contribution may be significant in some specific seaweeds. In some nori and wakame samples, a portion of 5 g could provide up to 70% of the allowed daily intake of cadmium. On the other hand, some kelp, wakame, and dulse samples presented a sizable contribution of lead, between 15–23%.

Concerning total arsenic, values obtained ranged from 0.4 mg/kg to 100 mg/kg. However, inorganic arsenic results were much lower in most of the samples. In nori, kelp, and sea spaghetti samples, inorganic arsenic could not be determined (< 0.10 mg/kg). The rest of the species other than hijiki had inorganic arsenic values close to the quantification limit, and their contribution to the TDI was not significant (<16%). Regarding hijiki, it must be highlighted the average of inorganic arsenic found was 62 mg/kg, much higher than the rest of species. This amount exceeds the corresponding TDI in up to 1780%, which means hijiki’s inorganic arsenic content may pose to a risk to consumers if ingested regularly.

Because both total arsenic and inorganic arsenic were quantified, it is possible to estimate the contribution of the inorganic fraction to the total. For hijiki seaweed, 70% of the total arsenic is in inorganic form, an overwhelming percentage compared to other species. This could lead to the conclusion that this species does not metabolize arsenic the same way than other types of seaweed. Moreover, for agar-agar, the inorganic arsenic fraction is also important (45%), but the amount of total arsenic is low compared to the rest of the species examined (only 0.4 mg/kg).

Finally, iodine contributions were extraordinarily high in most cases. Only sea lettuce, agar-agar, and nori sample contributions were lower than 100% (up to 57%, 61%, and 72%, respectively). On the other hand, a 5 g portion of kelp or kombu exceeded the tolerable daily intake by approximately 3000 and 4000%, respectively, which could mean that an important potential risk exists in consuming kelp and kombu. In addition, EFSA published afterwards that an adequate intake for adults was 150 μg/day, lower than the upper TDI used in this study, which suggests a worse picture for the risk assessment.

The method has proved to be suitable in analyzing heavy metals, arsenic, and iodine in dried seaweed simultaneously in a unique sample pretreatment, over- coming iodine determination issues using ICP-MS.

A 5 g daily portion of seaweeds like agar-agar, dulse, nori, sea spaghetti and wakame does not present a critical risk to consumers. However, a decent amount of cadmium, lead, and iodine may be found in some of them.

Sea lettuce contain the lowest values of metals, arsenic, and iodine. However, only one sample was included in this study, so clear conclusions cannot be reached.

Inorganic arsenic content in hijiki seaweed were extraordinarily high in contrast to other seaweeds. According to the results of this study, it is recommended that humans only consume 2.5 g per week of hijiki seaweed.

Kelp and kombu species were found to contain extremely high concentrations of iodine. Considering the results obtained, an intake of no more than 1 g per week of these seaweeds is suggested; regular consumption of higher amounts may lead to iodine excess disorders in consumers.

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(S3), e8727 (2020). DOI: 10.1002/rcm.8727.

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(23) EFSA (European Food Safety Authority), 

 is with the Head División at the Laboratorio de Salud Pública de Madrid Salud in Madrid, Spain. 

 is with the Technical Laboratory at the Laboratorio de Salud Pública de Madrid Salud in Madrid, Spain. 

 is an Assistant to the Department at the Laboratorio de Salud Pública de Madrid Salud in Madrid, Spain. Direct correspondence to: 

Seaweed has become a popular ingredient in Western diets. Concerns for heavy metals and inorganic arsenic have been raised as potential risks to consumers. Methods for analysis are described, including microwave digestion, HPLC,
and ICP-MS for quantitation and speciation.

Javier Peinador Asensio

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