Javier Peinador Asensio

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.

(1) A.H. Buschmann, C. Camus, J. Infante, A. Neori, A. Israel, M..C. Hernández-González, S.V. Pereda, J.L. Gómez-Pinchetti, A. Golberg, N. Tadmor-Shalev, and A.T. Critchley, 

(2) E.M. Brown, P.J. Allsopp, P.J. Magee, C.I. Gill, S. Nitecki, C.R. Strain, and E.M. Mcsorley, 

(4) P. MacArtain, C.I. R. Gill, M. Brooks, R. Campbell, and I.R. Rowland, 

(5) V. Quitral, C. Morales, M. Sepúlveda, and M. Schwartz, 

(6) J.L. Banach, E.F. Hoek-van den Hil, and H.J. van der Fels-Klerx, 

(8) Z. Ma, L. Lin, M. Wu, H. Yu, T. Shang, T. Zhang, and M. Zhao, 

(10) M. Leermarkers, W. Baeyens, M. De Gieter, B. Smedts, C. Meert, H.C. De Bisschop, R. Morabito, and P.H. Quevauviller, 

(11) Scientific Committee on Food (SCF), 

Scientific Panel on Dietetic Products, Nutrition and Allergies. Tolerable Upper Intake Levels for Vitamins and Minerals 

(2006). https://www.efsa.europa.eu/sites/default/files/efsa_rep/blobserver_assets/ndatolerableuil.pdf.

(12) M.B. Zimmerman, Y. Ito, S.Y. Hess, K. Fujieda and L. Molinari, 

(14) A. Lähteenmäki-Uutela, M. Rahikainen, M.T. Camarena-Gómez, J. Piiparinen, K. Spilling, and B. Yang, 

(15) Commission Regulation (EC) No 629/2008 of 2 July 2008 amending Regulation (EC) No 1881/2006 setting maximum levels for certain contaminants in foodstuffs. https://eur-lex.europa.eu/legal-content/EN/TXT/?uri=CELEX%3A32008R0629.

Commission Recommendation (EU) 2018/464 of 19 of March 2018 on the monitoring of metals and iodine in seaweed, halophytes and products based on seaweed.

 https://eur-lex.europa.eu/legal-content/EN/TXT/uri=uriserv%3AOJ.L_.2018.078.01.0016.01.ENG.

(17) M.S. Reid, K.S. Hoy, J.R.M. Schofield, J.S. Uppal, Y. Lin, X. Lu, H. Peng, X.C. Le, 

(18) H.J. Reid, A.A. Bashammakh, P.S. Goodall, M.R. Landon, C. O ́Connor, and B.L. Sharp, 

(19) E.M.M. Flores, P.A. Mello, S.R. Krzyaniak, V.H. Cauduro, and R.S. Picoloto, 

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

(20) K. Fujisaki, H. Matsumoto, Y. Shimokawa, and K. Kiyotaki, 

(22) M.Y. Roleda, H. Marfaing, N. Desnica, R. Jóns-dóttir, J. Skjermo, G. Rebours, and U. Nitschke, 

(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: 

Articles

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.

Studying Inorganic Arsenic, Heavy Metals, and Iodine in Dried Seaweed

This study analyzes the presence of inorganic arsenic and heavy metals in different varieties of rice cakes, both so-called "bio" (organic) and "non-bio," as well as those made with white rice and brown rice. Determination of cadmium (Cd), mercury (Hg), nickel (Ni), and lead (Pb) has been carried out by inductively coupled plasma mass spectrometry (ICP–MS) for samples previously digested in a microwave oven. The analysis of inorganic arsenic has been carried out by liquid chromatography (HPLC) combined with inductively coupled plasma–mass spectrometry (HPLC-ICP–MS) prior to acid extraction. All samples analyzed are below the limits established by local regulations. However, the content found for inorganic arsenic, cadmium, and nickel may be a risk to health for children, who are the marketing target for some of the rice cake products.

	Today, societal concerns about maintaining a healthy and balanced diet is causing an increase in the consumption of certain foodstuffs that are apparently healthier. It should be noted there is a growing interest among consumers about rice cakes, eaten as an appetizer or a snack replacement for more caloric foods, such as sweet cakes or snack chips. It is possible, however, that these rice products are not as healthy as they seem, because there are studies that demonstrate the presence of certain contaminants. In spite of this, a variety of rice cakes are marketed for consumption by the entire population, and in some cases intended specifically for infants and children.

	As a result of this trend toward consumption of healthy products, there is also a boom in the production and consumption of products categorized as organic, or 

products. For consumers, organic products are more popular, because they are considered to be of higher quality and believed to be free of possible contaminants that normal products may have.

	Numerous studies (1–5) have been published showing disturbing amounts of inorganic arsenic (iAs) in these products. In this regard, the national food agency of Sweden (National Food Agency, NFA) conducted a study (6) on the content of inorganic arsenic in rice products in 2015 and recommended that children under six years old should not be fed rice cakes, due to their high content of iAs.

	Arsenic is a metal that is found naturally in the Earth's crust; it is present in the air, water, and soil. In the case of food, it can occur in several different chemical forms, both organic and inorganic. Nowadays, there is scientific evidence demonstrating that inorganic arsenic, both As (III) and As (V), is the most toxic and harmful to health, because it has been proven that it can cause problems related to fetal development, neurotoxicity, diabetes, and cardiovascular diseases. In addition, the International Agency for Research on Cancer has classified it as "carcinogenic to humans" (7), because it can cause skin cancer, bladder cancer, and lung cancer.

	The scientific technical committee on contaminants in the food chain (Contam) of the European food safety authority (EFSA) adopted an opinion (8) on arsenic in food. It modifies the provisional tolerable weekly intake (ISTP), established by the Joint Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO) (FAO/WHO) Expert Committee on Food Additives (JECFA), based on the existence of studies demonstrating the toxicity of the iAs with exposure levels lower than those reviewed by JECFA. Thus, Contam set a limit of confidence between 0.3 and 8 µg/kg of body weight per day.

	Scientific opinion (8) established that children under three years of age were those most exposed to foods with iAs, because rice is a predominant ingredient in a wide range of foods for this group. In general, it is estimated that dietary exposure to iAs of infants and young children is between double and triple that of adults.

	In this context, this study has been carried out for the determination of iAs and expanded to included the analysis of various heavy metals, in order to conduct a more exhaustive study of this type of food. The specific heavy metals analyzed were cadmium, mercury, nickel, and lead.

	The concentration of these heavy metals can be considered especially relevant when dealing with toxic elements in doses that are harmful to the health of consumers. Except for Ni, these heavy metals are regulated, and in numerous scientific studies, their presence has been demonstrated in food and the consequences of their consumption have been studied.

	The European Union (EU) has approved Commission Regulation (EU) 2015/1006 (9), which modifies the Commission Regulation (CE) 1881/2006, in which the maximum content of iAs for rice and rice products is established. In a similar way, measures at the global level (CODEX Alimentarius) have also been adopted, so it is possible to carry out control measures of iAs in rice and products derived from rice.

	Commission Regulation (EU) 488/2014 (10), Commission Regulation (EU) 2015/1005 (11), and Commission Regulation (EU) 2018/73 (12) establish maximum limits for Cd, Pb, and Hg in foods, respectively. However, there are no limits in the case of Ni. Currently, European authorities are conducting a study (13) to establish maximum limits for this element in foodstuffs, as indicated in the Commission Recommendation (EU) 2016/1111 of 6 July 2016 on the monitoring of nickel in food (14).

	The samples analyzed in this study correspond to different varieties of rice cakes, both with the "bio" (organic) designation and without it, and rice cakes made with white and brown rice, all sold in the city of Madrid. In total, 29 samples of rice cakes were analyzed, of which 11 were labelled as "bio," while the remaining 18 did not have this designation. Note that 10 of the analyzed samples were made with white rice, and 19 with brown rice, because different studies have shown different amounts of iAs in white rice and brown rice.

	The samples were digested in a microwave oven (Milestone model Ethos One) equipped with output power regulation and automatic control systems for temperature and pressure of all the vessels, which allows for the monitoring of both parameters during the process. The containers used for samples are PTFE tubes with lids, with a capacity of 100 mL; they were previously subjected to a rigorous cleaning process, according to the manufacturer's instructions.

	The temperature program used for digestion is detailed in Table I (for the determination of heavy metals) and Table II (for the determination of iAs).

	For the analysis of heavy metals, an inductively coupled plasma–mass spectrometry (ICP–MS) instrument (Agilent Technologies model 7900), equipped with an 89 position integrated autosampler (IAS), was used. The operating parameters of the ICP–MS instrument are outlined in Table III.

	Inorganic arsenic (iAs) determinations were performed using a high performance liquid chromatography (HPLC) instrument (Agilent Technologies model 1200 Infinity), equipped with a model 1260 Quat Pump VL quaternary pump, an injector (model 1260 ALS), and a column compartment (model 1260 TCC). This HPLC system was used in conjunction with the ICP–MS instrument referred to above. The operating parameters of this system are listed in Table IV.

	The volumetric materials used for all solutions were calibrated polypropylene single-use tubes (DigiTubes SCP Science), except for the analysis of iAs, for which centrifuge tubes, also single-use, were used (VWR Metal Free, 15 mL volume). Eppendorf brand micropipettes were used for sampling corresponding aliquots.

	For the preparation of the reagents and solutions for washing and rinsing of the work material, deionized water (H

O) class type I (resistivity: 18.2 MO cm) was used, purified using Milli-Q Integral system. The nitric acid (HNO

, 65%) and hydrochloric acid (HCl, 37%) utilized are quality pro analysis (p.a.), supplied by Scharlau, and distilled in the laboratory by a Savillex distiller model DST-1000. The hydrogen peroxide (H

) was from Merck, 30%, Suprapur. Glacial acetic acid was from Scharlau, 99.8%, ultratrace and ppb-trace analysis grade. The tuning solution for the ICP–MS instrument (Agilent Technologies) contained 1 µg/L Ce, Co, Li, Mg, Tl and Y. Finally, the argon and helium (99.999%) used in plasma generation and interference elimination were supplied by CONTSE.

	In the case of the determination of heavy metals, all standard solutions were prepared with a mixture of 2% HNO

 and 1% HCl, based on the following standards, having concentrations of 1000 mg/L: Cd (Sigma-Aldrich), and Hg, Ni and Pb (VHG-Labs).

	Intermediate solutions of elements of interest were prepared as follows: Cd (1 mg/L), Hg (1 mg/L), Ni (10 mg/L), and Pb (1 mg/L) were used to prepare a multielemental standard (composed of 4 µg/L Cd, 5 µg/L Hg, 50 µg/L Ni, and 10 µg/L Pb) which is later used to prepare the calibration standards. To do this, the standard is taken and diluted 100 times, 25 times, 10 times, and 2.5 times, to obtain a calibration function with 5 levels.

	For validation of Cd, Ni, and Pb, a multi-element standard of 1 µg/L was prepared from a commercial standard of 100 mg/L, provided by Scharlau, and a standard of 1.016 µg/L Hg was prepared from a 0.1016 mg/L NIST reference material, used for verification of the Hg control. Finally, 400 µg/L of Ir and Rh, prepared in a solution with 2% HNO

, 1% HCl, and 5% acetic acid, was used as the internal standard solution. This internal standard was prepared from solutions of 1000 mg/L of Ir (Sigma-Aldrich) and Rh (Panreac).

	For the analysis of inorganic arsenic, the solution used for extraction is 0.2% HNO

, noting that the solution used for the preparation of all standards was 0.2% HNO

	For inorganic As, the calibration standards were prepared from a standard of As (V) (from As2O5) of 100 mg/L in H

O (VHG Labs). Using an intermediate solution of 1 mg/L standard with 10 µg/L prepared, this ssolution was diluted 2.5 times, 10 times, 25 times and 50 times, to obtain a calibration function with 5 levels.

	An analysis of another As (V) reference material of was included, with the same specifications as the previous one, but in a different batch. Furthermore, a control of the process of oxidation of As (III) to As (V), using a standard of As (III), supplied by VHG Labs (from As

	The mobile phase composition was 20 mM PBS / 2 mM EDTA / 3% MeOH, in deionized water class type I, pH = 6, using Panreac methanol (CH

OH) with a minimum assay of 99.9%, pure Panreac disodium ethylenediaminetetraacetate dihydrate (EDTA, with an assay of 99.0–101.0%, and pure Panreac sodium dihydrogen phosphate dihydrated (assay 98.0–100.5%).

	The steps of the analytical process are shown in Figure 1.


	Figure 1: The steps in the analytical method used in this study.

	The homogenization of all samples was achieved by following the method described in the standard UNE-EN 13804:2013 (15), using a grinder to obtain a representative sample of the product to be tested.

	To begin, the homogenized samples were weighed to 0.50 ±0.05 g. For the digestion process, 3 mL HNO

 were added and digested in the microwave (conditions described in Table I), and, once the process was complete, samples were left to cool down to room temperature. The solutions were then transferred to the DigiTubes filled with up to 50 mL deionized water. These prepared samples were then ready to be analyzed by ICP–MS.

	In this study, the determination of inorganic arsenic was carried out as the sum of arsenite (As [III]) and arsenate (As [V]). This quantifies the peak corresponding to the As (V) obtained with a retention time of 4.75 min (see Figure 2).


	Figure 2: Arsenic speciation chromatogram showing a peak at 4.75 min for arsenate (As V).

	The homogenized sample has been weighed 0.2 ±0.02 g, and 10 mL of the solution prepared for extraction has been added. Later, the sample was treated in the microwave (conditions described in Table II). In this process, all of the As (III) in the sample is oxidized to As (V). Once the process has been completed, samples are left to cool down to ambient temperature, transferred to 15 mL tubes, and centrifuged for 10 min at ≥4000 rotations per min (rpm) The supernatant is filtered using 0.45 µm pore size nylon filters, and is poured into 1 mL polypropylene vials to inject into the HPLC instrument.

	The analytical method used for the determination of heavy metals is required to meet the criteria of the Commission Regulation (EC) N° 333/2007 of March 28, 2007 (16) laying down the methods for sampling and analysis for the official control of the levels of lead, cadmium, mercury, inorganic tin, 3-monochloropropanediol (3-MCPD), and benzo(a)pyrene in foodstuffs and its modifications. Validation has been carried out according to the requirements of the standard UNE/EN-ISO 17025 (17) for testing laboratories, and it is accredited by the Entity National Accreditation (ENAC). In the case of the determination of iAs, the analytical method is in process of accreditation. The results of the validation are shown in Table VI.

	For quality control, we have participated in different intercomparison exercises and additions of known concentrations of the analyte used to assess the accuracy of the method. In the case of iAs, a standard of As (III) has been used as described in Section 2.2.

	The results obtained in this study are shown in Table V, detailing the maximum concentrations and average concentrations obtained for iAs, Cd, Ni, and Pb, as well as the number of samples that have been quantified in each case. The results for Hg are not included in this table, because the concentrations obtained are <0.010 mg/kg, and they have not, therefore, been able to be accurately quantified.

	All the results obtained in the determination of iAs were able to be quantified. Nevertheless, it should be noted that no sample exceeded the maximum value established by Commission Regulation (EU) 2015/1006 (9) regarding maximum levels of inorganic arsenic in foodstuffs, set at 0.30 mg/kg for rice cakes. However, taking into account a value of 0.3 µg/kg of body weight as the threshold of tolerable risk considered in the studies carried out by the EFSA (8), in the case of a six-year-old child (with an approximate weight of 20 kg), for example, the admissible threshold would be 6 µg of iAs, and the consumption of a single rice cake (about 30 g), with the highest value of found iAs (0.256 mg/kg) would result in the intake of approximately 7.7 µg of iAs. Therefore, special attention should be given to rice cake consumption by children.

	In the case of Pb, the results obtained are not considered significant, because the concentrations were below the thresholds referred to in toxicity studies published for this heavy metal (18).

	In terms of the results obtained for Cd, 60% of the analyzed samples are quantified as having a concentration >0.010 mg/kg. It should be noted that all of these results are below the reference value in the Commission Regulation (EU) 488/2014 (10), where the maximum content of Cd in rice has been established as 0.20 mg/kg. However, health authorities establishes a tolerable daily intake of 0.36 µg/kg body weight (19) of this heavy metal to ensure all consumers have sufficient protection. Referring to a previous example, a six-year-old child should not exceed 7.1 µg of Cd daily intake. Hence, based on the data obtained in this study and where the maximum found value was present (0.181 mg/kg), consumption of a single rice cake would contribute 5.4 µg of Cd.

	On the other hand, the values obtained for Ni show a high concentration of this metal in rice cakes, levels that even exceeded the concentrations of other contaminants that are regulated. Taking as a reference the measured maximum value, the consumption of a single rice cake would contribute up to 67.8 µg of Ni, which, at first sight, could be considered relevant if compared to the established maximum levels for other metals.

	In addition, in the case of iAs, there are no significant differences between the rice cakes considered to be "bio" and "non bio"; on the other hand, more iAs is seen in the rice cakes made with brown rice than those made with white rice. The content of iAs is not affected by the type of rice cultivation (bio or non bio), but the processing of the rice has important influence as a factor that reduces the concentration of iAs in white rice compared to brown rice, as shown in Figure 3.


	Figure 3: Results obtained in the study for cadmium, nickel, and inorganic arsenic (iAs). BIO: Total of analysed samples of rice cakes "BIO" (Brown + White); Non BIO: Total of analysed samples of rice cakes "Non BIO" (Brown + White); WHITE: Total of analysed samples of rice cakes "White rice" (BIO + Non BIO); BROWN: Total of analysed samples of rice cakes "Brown rice" (BIO + Non BIO).

	The results for Cd and Ni highlight that the "bio" rice cakes present concentrations clearly higher than the "non-bio" rice cakes, as shown in Figure 3. In the case of Cd, there are also significant differences between rice cakes made with white rice versus those made with brown rice. On the contrary, in the case of Ni, we can see that there are no significant differences between the content of these metals in either variety of cake.

	In this case, based on the determined quantities of Cd and Ni, we can appreciate a large influence in the type of rice cultivation but not so much in the processing of the rice.

	All rice cakes samples analyzed comply with the legislation in force, because they do not exceed the maximum levels permitted; therefore, these foods should be considered safe for consumption.

	However, based on the results obtained in this study for iAs, Cd, and Ni, it would be advisable that consumption of rice cakes by children be regulated by health authorities and that greater control be maintained over rice cakes that have with both the designation of "bio" and are made from brown rice.

	On the other hand, the test method used for the analysis of Cd, Hg, Ni, and Pb by microwave acid digestion and determination by inductively coupled plasma–mass spectrometry is considered a valid option to carry out a study as presented in this publication, obtaining results in accordance with the requirements of the UNE-EN ISO 17025 standard.

	The coupling of liquid chromatography to ICP–MS is a recommended method for chromatographic separation of the different species of arsenic, for the analysis of inorganic arsenic, determined as the sum of As (III) and As (V).

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(6), 1164-1178 (2018). DOI: 10.1080/19440049.2018.1440641.

	(2) A.J. Signes-Pastor, M. Carey, and A.A. Meharg, 

	(3) M. Molin, S.M. Ulven, H. Margrete Meltzer, and J. Alexander, 

	(4) H.N. Lynch, G.I. Greenberg, M.C. Pollock, and A.S. Lewis, 

	(5) N. Ferré-Huguet, R. Martí-Cid, M. Schuhmacher, and J.L. Domingo, 

	(6) Livsmedelsverket National Food Agency, Sweden. 2015. Study reveals problems with arsenic in rice and rice products. 

https://www.livsmedelsverket.se/en/about-us/press/study-reveals-problems-with-arsenic-in-rice-and-rice-products

	(7) International Agency for Research on Cancer (IARC). Monographs on the Identification of Carcinogenic Hazards to Humans. List of classifications, volumes 1-123. 

https://monographs.iarc.fr/list-of-classifications-volumes/

	(8) EFSA (European Food Safety Authority): EFSA Panel on Contaminants in the Food Chain (CONTAM): Scientific Opinion on Arsenic in Food. EFSA Journal 7(10), 1351 (2009).

	(9) Commission Regulation (EU) 2015/1006 of 25 June 2015 amending Regulation (EC) No 1881/2006 as regards maximum levels of inorganic arsenic in foodstuffs.

	(10) Commission Regulation (EU) No 488/2014 of 12 May 2014 amending Regulation (EC) No 1881/2006 as regards maximum levels of cadmium in foodstuffs.

	(11) Commission Regulation (EU) 2015/1005 of 25 June 2015 amending Regulation (EC) No 1881/2006 as regards maximum levels of lead in certain foodstuffs.

	(12) 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.

	(13) EFSA (European Food Safety Authority): EFSA Panel on Contaminants in the Food Chain (CONTAM): Scientific Opinion on the risks to public health related to the presence of nickel in food and drinking water. EFSA Journal 

	(14) Commission Recommendation (EU) 2016/1111 of 6 July 2016 on the monitoring of nickel in food.

	(15) UNE-EN 13804:2013. Foodstuffs - Determination of elements and their chemical species - General considerations and specific requirements.

	(16) Commission Regulation (EC) No 333/2007 of 28 March 2007 laying down the methods of sampling and analysis for the official control of the levels of lead, cadmium, mercury, inorganic tin, 3-MCPD and benzo(a)pyrene in foodstuffs.

	(17) General requirements for the competence of testing and calibration laboratories (ISO/IEC 17025:2017).

	(18) EFSA (European Food Safety Authority): EFSA Panel on Contaminants in the Food Chain (CONTAM): Scientific Opinion on Lead in Food. EFSA Journal 

	(19) EFSA (European Food Safety Authority). 2012. Scientific Report: Cadmium dietary exposure in the European population. 

Javier Peinador Asensio, Silvia Borobia Caminos, 

 are with the Public Health Laboratory of Madrid, Spain. Direct correspondence to: 

Study of the Content of Inorganic Arsenic and Heavy Metals in Samples of Rice Cakes in the City of Madrid