Human Exposure to Toxic Metals (Cd, Pb, Hg) and Nitrates (NO₃⁻) from Seaweed Consumption

Abstract

Seaweed is now considered a functional food with a high nutritional value in Western countries, and the consumption of different species of edible algae has grown exponentially in recent decades. However, anthropogenic pressure on the seas has increased the presence of pollutants such as toxic metals and nitrates that can accumulate in algae. It is necessary to know the levels of these contaminants and the dietary exposure from the consumption of edible algae. The content of toxic metals (Cd, Pb, Hg) and nitrates (NO₃⁻) was determined in 72 samples of edible algae marketed in the Canary Islands (Spain). Cd stands out in the Asian algae hijiki (1.196 mg/kg) and nori (1.005 mg/kg). Pb stands out in the Asian wakame seaweed (0.119 mg/kg). The highest mean concentration of Hg was in European arame algae (0.055 mg/kg). Some samples of the nori seaweed had NO₃⁻ concentrations of >8000 mg/kg. Considering the consumption recommended by the manufacturer of 4 g/day, the maximum admissible intake values are not exceeded, and, consequently, this does not pose a risk to health. However, in the case of Cd, Pb, Hg, and NO₃⁻, legislation is necessary to regulate their content in edible algae.

1. Introduction

The consumption of seaweed has long been part of the traditional diet in Asia and is currently growing in popularity in the Western world. According to the Food and Agriculture Organization of the United Nations (FAO), algae production in Europe accounts for 10% of global production [1,2,3]. Edible algae can be classified into four main large groups: blue, red, brown, and green algae.

Algae stand out for their vitamin B₁₂, omega-3 fatty acids, selenium, iodine, and fiber content [4]. However, in addition to high-value nutrients, they are capable of transferring toxic metals such as cadmium (Cd), lead (Pb), or mercury (Hg) to the consumer. These elements of toxicological relevance are present in marine waters as pollutants, which are characterized by their bioaccumulation in the trophic chain, or nitrates (NO₃⁻) from both natural sources (nitrogen cycle) and anthropogenic sources (fertilizers, intensive livestock, wastewater, etc.) [5,6,7].

Cd is a metal that induces tissue damage due to oxidative stress. It is nephrotoxic as it damages the renal tubule; it can cause deterioration of mitochondrial function, and because of its divalent nature, it can compete with essential elements such as Zn [8,9]. The consumption of marine products such as algae, fish, or shellfish is an important source of exposure to Cd [10,11]. The European Food Safety Authority (EFSA) has set a tolerable weekly intake (TWI) for Cd of 2.5 µg/kg person/week [10].

Pb is one of the most toxic metals known; it is considered carcinogenic (Group 2B) for humans [12]. In adults, it causes cardiovascular, central nervous system, kidney, and fertility problems [13]. It is found in high concentrations in fruits, vegetables, and cereals as a result of the deposition of atmospheric Pb [14,15]. EFSA has established a benchmark dose (BMDL) value referring to the organospecific toxicity of Pb of 0.63 µg/kg body weight (bw)/day (nephrotoxic effects) and 1.5 µg/kg bw/day (cardiovascular effects) [16].

Hg can be found in the form of different chemical compounds of toxicological relevance [17]. Methylmercury compounds can cause brain damage, psychological disorders, deafness, loss of vision and motor skills, etc. Inorganic mercury compounds can damage the kidneys, liver, and brain [18]. EFSA has set TWI values of 4 μg/kg bw/week (inorganic Hg) and 1.3 μg/kg bw/week (methylmercury) [18].

Nitrates (NO₃⁻ are found naturally in the environment. However, the concentration of these anions has increased as a result of human activity (use of fertilizers, intensive livestock farming, household waste, etc.). Nitrates, once in the body, can be reduced to nitrites by bacterial action and can then promote the formation of N-nitroso compounds, classified as carcinogens by the International Agency for Research on Cancer (IARC) [19]. Nitrates and nitrites are classified by IARC as “probable human carcinogens” (Group 2A) under certain conditions [20].

Scientific evidence associates a high exposure to nitrates with an increase in the incidence of gastric, colorectal, esophageal, thyroid, and kidney cancer, among others [6,21]. Likewise, especially in the case of children, a high intake of these anions is related to blue baby syndrome or methemoglobinemia [6,22].

Algae are autotrophic organisms that perform photosynthesis; that is, by their very nature, they use nitrogen and its derivatives for their biological cycles. Therefore, these organisms concentrate higher concentrations of nitrates. Despite this, algae are a food that, to date, has not been assigned legal nitrate limits, and this poses a risk for consumers.

The World Health Organization (WHO) and EFSA set an acceptable daily intake (ADI) of 3.7 mg/kg bw/day for nitrates [23], which remains in force today, despite recent re-evaluations by EFSA [24,25].

Bearing in mind the above, there is a clear need to determine the content of toxic metals (Cd, Pb, Hg) and nitrates in the different species of edible algae marketed in Europe in order to evaluate the contribution of their consumption to permitted intakes and thereby determine the safety of these products.

2. Material and Methods

All chemicals and materials used were of analytical quality or equivalent and were the following: hydrogen peroxide (H₂O₂) (Sigma Aldrich, Darmstadt, Germany), nitric acid (HNO₃) of 65% purity (Sigma Aldrich, Germany), sodium nitrate (NaNO₃) (99%) purity (Panreac, Spain), disodium tetraborate (Na₂B₄O₇·10H₂O) (Sigma Aldrich, Darmstadt, Germany), dibasic phosphate (Na₂HPO₄) (Merck, Darmstadt, Germany), LC-MS grade methanol (CH₃OH) (Panreac, Barcelona, Spain), potassium dihydrogen phosphate (H₂KO₄P) (Panreac, Spain), and C18 solid-phase extraction cartridges (6 mL, 500 mg) (Waters, Milford, MA, USA). The solutions were prepared with purified water from a Milli-Q Plus system (Millipore, Burlington, MA, USA).

2.1. Samples

A total of 72 samples of edible algae (green, brown, and red) marketed in Tenerife (Canary Islands) and acquired in different commercial areas between the months of January and December 2020 were analyzed (

Table 1. Characteristics of the analyzed algae samples.

Species	Common Name	Type	No. Samples	Origin
Gracilaria	Agar agar	Red	3	Unknown
Undaria pinnatifida	Wakame	Brown	5	Galicia (Spain, EU)
			4	Japan (Non-EU)
			3	Unknown
Laminaria ochroleuca	Kombu	Brown	13	Galicia (Spain, EU)
			3	Japan (Non-EU)
			3	Unknown
Sargassum fusiforme	Hijiki	Brown	3	Japan (Non-EU)
Porphyra	Nori	Red	5	Korea (Non-EU)
			3	Japan (Non-EU)
			4	Galicia (Spain, EU)
Eisenia bicyclis	Arame	Brown	4	Japan (Non-EU)
Himanthalia elongata	Sea spaghetti	Brown	7	Galicia (Spain, EU)
Ulva Lactuca	Sea lettuce	Green	4	Galicia (Spain, EU)
Mastocarpus stellatus	Starry moss	Red	4	Galicia (Spain, EU)
-	Mixed salad	Brown and red	4	Galicia (Spain, EU)

).

The samples were stored at room temperature and in their original containers until treatment. The analyzed algae belong to the species Eisenia bicyclis (Arame), Laminaria ochroleuca (Kombu), Undaria pinnatifida (Wakame), Gracilaria (Agar agar), Sargassum fusiforme (Hijiki), Porphyra (Nori), Himanthalia elongata (Sea spaghetti), Ulva Lactuca (Sea lettuce), Mastocarpus stellatus (Starry moss), and mixed salad.

In the case of the determination of nitrates, the sampling was planned according to the criteria established in Regulation (EC) No. 1882/2006 of the Commission, which sets out the sampling requirements and analytical methods for the official control of the content of nitrates in certain food products [26].

2.2. Treatment and Determination of Toxic Metals

One gram of each previously homogenized sample was introduced into Teflon digestion vessels (GO for Smart Vent, Anton Parr, Austria) and made up to 65% with 2 mL of hydrogen peroxide (H₂O₂) (Sigma Aldrich, Darmstadt, Germany) and 4 mL of nitric acid (HNO₃) (Sigma Aldrich, Darmstadt, Germany). The Teflon digestion vessels were closed, and the digestion was started in a microwave oven (Multiwave GO Plus, Anton Parr, Austria), applying the digestion program shown in

Table 2. Instrumental conditions of the microwave digestion process.

No.	Ramp (min)	Temperature (°C)	Time (min)
1	15′00″	50	5′00″
2	5′00″	60	4′00″
3	5′00″	70	3′00″
4	3′00″	90	2′00″
5	20′00″	180	10′00″

Temperature limit: 200 °C. Cooling limit: 50 °C.

. Three replicates were made for each analyzed sample.

After digestion, the samples were transferred to a 10 mL volumetric flask and filled up to the mark with Milli-Q quality distilled water. The samples were then deposited in airtight jars with labeled lids for measurement.

The analytical method was atomic absorption spectrophotometry (AAS) [27]. AAS is an analytical method approved in Commission Regulation 333/2007 (EC) of 28 March 2007, which sets out the sampling and analysis methods for the official control of the levels of lead, cadmium, mercury, inorganic tin, 3-MCPDF, and benzo (a)pyrene in food products [28], which was later modified by Regulation 836/2011 [29].

The determination of Cd and Pb was performed with an atomic absorption spectrophotometer (AS-800, PerkinElmer, Waltham, MA, USA) with a graphite chamber (HGA-800, Perkin Elmer, USA) (GF-AAS). The Hg determination was carried out with a cold vapor atomic absorption spectrophotometer (AS-800, PerkinElmer, Waltham, MA, USA) (CV-AAS) with a flow injection system (FIMS-400, PerkinElmer, Waltham, MA, USA). The instrumental wavelengths (nm) were Cd (228.8), Pb (283.3), and Hg (253.7). The instrumental limits of quantification (LOQ) of the method were Cd (0.020 mg/kg), Pb (0.040 mg/kg), and Hg (0.10 mg/kg).

Table 3. Instrumental conditions of the graphite chamber for the determination of Cd and Pb and of the cold vapor for the determination of Hg.

Cd	Step	Temp. (°C)	Ramp time (min)	Hold time (min)	Internal Flow	Gas type
	1	110	10	20	250	Normal
	2	130	15	30	250
	3	700	10	20	250
	4	1500	0	5	0
	5	2450	1	3	250
Pb	Step	Temp. (°C)	Ramp time (min)	Hold time (min)	Internal Flow	Gas type
	1	110	1	30	250	Normal
	2	130	15	30	250
	3	700	10	20	250
	4	1500	0	5	0
	5	2450	1	3	250
	Read step: 4; Injection temp. (°C): 20
	Volume: 20 µL		Diluent volume: 0 µL		Diluent location: 131
Hg	Time (s)	Pump 1 speed	Pump 2 speed		Valve position
	15	100	120		Fill
	11	100	120
	15	0	120		Inject

shows the instrumental conditions for the determinations of Cd, Pb, and Hg.

The quality control of the method to ensure the precision and accuracy of the analytical procedure was based on the recovery study with certified reference material under reproducible conditions. The reference material used was as follows: Cd and Pb (BCR-279 Sea Lettuce, British Certified Reference) and Hg (NIST SRM 1577 BL, National Institute of Standards and Technology; BCR-278 R MT). In all cases, recovery percentages greater than 95% were obtained.

2.3. Treatment and Determination of Nitrates

The entire analytical procedure for the determination of nitrates in algae was performed according to the official method EN 12014-4: 2005 and was carried out at the Public Health Laboratory of Las Palmas de Gran Canaria, which is accredited with the ISO/IEC 17,025 standard. The method is accredited by the Spanish National Accreditation Entity (ENAC).

A 100 g portion of the sample (edible part) was homogenized using a laboratory grinder (Knifetc 1095, Foss, Hillerød, Denmark), after which 2.5 ± 0.2 g of homogenized sample were mixed with 25 mL of ultrapure water at 70 °C and 1.25 mL of a saturated solution of disodium tetraborate. After mixing, the samples were boiled for 15 min in a thermostatted bath (Precisterm 6000141, Selecta, Madrid, Spain). After cooling, ultrapure water was added to the mixture to a weight of 61.5 g and then centrifuged at 4500 rpm for 15 min (Macrotronic BL centrifuge, Selecta, Madrid, Spain). The supernatant was cleaned using C18 cartridges that were preconditioned with 5 mL of methanol.

The purified extracts were diluted 1:1 with ultrapure water before chromatographic analysis. Based on previous laboratory experience, the samples that were expected to have a high nitrate concentration (>625 mg/kg) were diluted at 1:20 with ultrapure water to match the measurement range.

Before placing the samples in the chromatography vials, the extracts were filtered through 0.2 µm × 47 mm syringe filters (VWR, Radnor, PA, USA). Chromatographic analysis was performed on the day of extraction [7].

The extracts were subjected to chromatographic analysis for quantitative determination using a Waters 2695 system (Waters Corporation, Milford, MA, USA), consisting of a binary pump, a photodiode array detector (PDA), and a Rheodyne injection valve with an injection loop of 25 μL. The system was interconnected to a personal computer for instrumentation control, data acquisition, and processing and equipped with chromatographic software (Empower ™, Waters). The chromatographic column was an anion IC-Pak (50 mm × 4.6 mm ID, 10 μm particle size). The injection volume was 10 µL. The mobile phases consisted of 0.05 M phosphate buffer (A) and ultrapure water (B) and were performed in an isocratic mode, 1.0 mL/min at a constant temperature (25 °C).

The PDA detector was programmed to a fixed wavelength of 210 nm. The nitrate retention time was 11.51 min, and the limit of quantification was 500 mg/kg. The relative measurement uncertainty within the entire validated range was 14%. The nitrate recovery percentage was 96.7–105.4% (low range: 500 mg/kg, 96.7%; mid-range I: 2000 mg/kg, 105.4%; mid-range II: 5000 mg/kg, 105.3%; high range: 8000 mg/kg, 103.2%).

2.4. Statistical Analysis

The computer software GraphPad 8.4.3 (GraphPad, San Diego, CA, USA) was used to perform the statistical analysis of the results.

A set of tests were applied to verify the normality of the analyzed data (Anderson–Darling, D’Agostino and Pearson, Shapiro–Wilk, and Kolmogorov–Smirnov). Once the aforementioned tests were applied, the data obtained were checked to see whether they followed a normal distribution; if they did not, non-parametric tests were applied, in this case, the Mann–Whitney test (two-tailed). Significant differences are considered when the value of p < 0.05.

2.5. Calculation of Dietary Intake

The evaluation of dietary intake is based on the previous calculation of the estimated daily intake (EDI) (Equation (1)). Its percentage contribution is then obtained taking the reference values of tolerable daily/weekly intake [30,31,32].

$$\mathrm{EDI}=\frac{\mathrm{Nitrate} \mathrm{or} \mathrm{Metal} \mathrm{concentration} \left(\mathrm{mg}/\mathrm{kg}\right)}{\mathrm{Mean} \mathrm{consumption} \left(\mathrm{kg}/\mathrm{day}\right)}$$

(1)

The mean average consumption of dehydrated seaweed, as stated by the manufacturers, is set at 4 g/day [33]. The mean average weight of a Spanish adult is 68.48 kg [34].

$$\mathrm{Contribution}\left(\%\right)=\frac{\mathrm{EDI} \left(\frac{\mathrm{mg}}{\mathrm{day}}\right)}{\mathrm{Reference} \mathrm{value} \left(\frac{\mathrm{mg}}{\mathrm{day}}\right)}\times 100$$

(2)

2.6. Calculation of the MoE

The Margin of Exposure (MoE) was calculated (Equation (3)) to determine the margin of exposure for non-carcinogenic effects. “E” is the exposure level, given by the amount of toxic metal or nitrate ingested (mg/day). NOAEL is the No Observed Adverse Effect Level. The BMDL is the Benchmark Dose Level.

$$\mathrm{MoE}=\frac{\mathrm{NOAEL} \mathrm{or} \mathrm{BMDL}}{\mathrm{E}}$$

(3)

3. Results and Discussion

3.1. Concentration and Dietary Intake of Toxic Metals (Cd, Pb, Hg)

Table 4. Cadmium concentrations, estimated intake (EDI), and percentage contribution to the guideline value of the analyzed algae.

Origin	Species	Concentration ± SD (mg/kg)	EDI (µg/day)	Contribution to TWI (%)
Asia	Hijiki	1.196 ± 0.234	4.78	19.6
	Nori	1.005 ± 0.897	4.02	16.4
	Kombu	0.417 ± 0.484	1.67	6.82
	Wakame	0.753 ± 0.463	3.01	12.3
Europe	Sea spaghetti	0.020 ± 0.032	0.08	0.33
	Kombu	0.085 ± 0.096	0.34	1.39
	Sea lettuce	<LOQ	-
	Starry moss	<LOQ
	Nori	0.011 ± 0.00	0.044	0.18
	Wakame	0.070 ± 0.096	0.28	1.14
	Arame	0.082 ± 0.125	0.328	1.34
	Mixed salad	0.198 ± 0.137	0.792	3.24

TWI, tolerable weekly intake.

,

Table 5. Lead concentrations, estimated intake (EDI), and percentage contribution to the guideline value of the analyzed algae.

Origin	Species	Concentration ± SD (mg/kg)	EDI (µg/day)	Contribution to BMDL (%)
				Pb (Np.)	Pb (Card.)
Asia	Hijiki	0.004 ± 0.001	0.016	0.04	0.02
	Nori	0.018 ± 0.019	0.072	0.17	0.07
	Kombu	0.071 ± 0.070	0.284	0.66	0.28
	Wakame	0.119 ± 0.071	0.476	1.10	0.46
Europe	Sea spaghetti	0.020 ± 0.032	0.08	0.19	0.08
	Kombu	0.041 ± 0.077	0.164	0.38	0.16
	Sea lettuce	<LOQ	-
	Starry moss	<LOQ
	Nori	0.011 ± 0.00	0.044	0.10	0.04
	Wakame	0.007 ± 0.007	0.028	0.06	0.03
	Arame	0.047 ± 0.008	0.188	0.44	0.18
	Mixed salad	0.016 ± 0.013	0.064	0.15	0.06

Np., nephrotoxicity; Card., cardiotoxicity; BMDL, benchmark dose level.

and

Table 6. Mercury concentrations, estimated intake (EDI), and percentage contribution to the guideline value of the analyzed algae.

Origin	Species	Concentration ± SD (mg/kg)	EDI (µg/day)	Contribution to TWI (%)
				Hg (Org.)	Hg (Inorg.)
Asia	Hijiki	0.017 ± 0.004	0.068	0.54	0.17
	Nori	0.010 ± 0.004	0.04	0.31	0.10
	Kombu	0.054 ± 0.017	0.216	1.70	0.55
	Wakame	0.021 ± 0.01	0.084	0.66	0.21
Europe	Sea spaghetti	0.007 ± 0.003	0.028	0.22	0.07
	Kombu	0.017 ± 0.011	0.068	0.54	0.17
	Sea lettuce	<LOQ
	Starry moss	0.003 ± 0.00	0.012	0.09	0.03
	Nori	0.003 ± 0.00	0.012	0.09	0.03
	Wakame	0.008 ± 0.008	0.032	0.25	0.08
	Arame	0.055 ± 0.003	0.22	1.73	0.56
	Mixed salad	0.004 ± 0.005	0.016	0.13	0.04

Org., organic mercury; Inorg., inorganic mercury; TWI, tolerable weekly intake.

show the mean concentrations (mg/kg dry weight/dw), standard deviations (SD), and the dietary intake assessment of the toxic metals (Cd, Pb, Hg) in the analyzed algae.

Cd is noteworthy for its high concentrations, and its content in Asian algae is worth mentioning, especially in hijiki (1.196 mg/kg) and nori (1.005 mg/kg) (Table 4). According to the EFSA, the Cd levels in some seafoods are remarkable. The levels of Cd in oysters (0.2350 mg/kg), bivalve mollusks (0.163 mg/kg), and limpets (0.180 mg/kg) stand out [10]. However, the Cd concentrations recorded in some seaweed samples are higher than the average Cd levels in some seafoods reported by EFSA.

Regarding Pb, the highest concentrations obtained also correspond to algae from Asian countries, in this case, wakame algae (0.119 mg/kg) (Table 5). The case of Hg is not the same since the highest mean average content was found in European algae, arame algae (0.055 mg/kg) (Table 6).

Figure 1 shows the comparison of the content of the three analyzed metals between Europe and Asia. It can clearly be seen that, in all cases, the concentrations of the three toxic metals are higher in Asia than in Europe (without differentiating between species). In addition, the statistical study revealed the existence of significant differences (p < 0.05) in the content of these metals between both origins.

The above-mentioned differences in the content of toxic metals between origins are due to the fact that, according to various studies, the high levels of contamination in toxic metals existing in the Asian coasts influence the quantity of these toxic metals in the environment where the algae grow. Notably higher levels of Cd, Pb, and Hg than those recorded in the present study have been reported in many areas of the Chinese coast [35].

The import of edible algae from Asian countries may pose a risk to the health of consumers in terms of toxic metal content. The environmental impact of toxic industrial discharges in algae cultivation areas such as the coast of Palk Bay in India [36] or the Gulf of Kutch in India [37] is of concern due to the increase in levels of heavy metals in algae. In many cases, an increase in Cd and Pb levels has been quantified in algae cultivated on the shores of Palk Bay, presenting levels of 4 mg Cd/kg dw (Caulerpa racemosa species) or up to 15 mg Pb/kg dw (Caulerpa racemosa species).

Figure 2 shows the mean concentrations of toxic metals (Cd, Pb, Hg) in the different species of analyzed algae. The differences found in the metal content between species are due to the intrinsic features of each species, with some species being more prone to accumulating some metals than others. Similarly, the statistical study confirmed the existence of significant differences (p < 0.05) in the content of Cd (arame vs. nori; sea spaghetti vs. hijiki, kombu, nori, mixed salad, and wakame; hijiki vs. kombu, mixed salad, and wakame; kombu vs. nori and wakame; nori vs. mixed salad and wakame), in the Pb content (arame vs. nori and mixed salad; sea spaghetti vs. wakame; hijiki vs. wakame; kombu vs. wakame; nori vs. wakame), and in the Hg content (arame vs. sea spaghetti, kombu, nori, mixed salad, and wakame; sea spaghetti vs. kombu, hijiki, and wakame; hijiki vs. nori and mixed salad; kombu vs. nori and mixed salad; nori vs. mixed salad and wakame).

A study conducted by Paz et al. [27] reported a Cd content in Asian wakame algae of 1.11 mg/kg, similar to the Cd content found in the present study in hijiki algae. In contrast, the Pb concentrations recorded by Paz et al. [27] were higher than those found in the study here. However, the Hg content recorded in the present work is higher than the data published by Paz et al. [27].

The recommended consumption of 4 g/day, according to the manufacturer on the packaging, of hijiki and nori algae accounts for a percentage contribution to the TWI of Cd (2.5 µg/kg bw/week) of 19.6% and 16.4%, respectively (Table 4). Although this percentage does not exceed the TWI, it should be borne in mind that it is nearly a quarter of this maximum value and that the global diet may have a high total intake of Cd. The MoE value calculated by the NOAEL (0.01 mg/kg bw/day) [38] is 2.1 (hijiki algae) and 2.5 (nori algae). Considering this consumption and the MoE values, there is no risk.

The consumption of wakame algae (4 g/day) is 1.10% of the BMDL of Pb set at 0.63 µg/kg bw/day (nephrotoxic effects) (Table 5) [16]. This percentage does not represent a significant contribution to the dietary intake of Pb, and therefore, adult consumers are not at risk. The MoE value calculated considering a BMDL₀₁ of 0.0015 mg/kg bw/day [16] is 3.15 (wakame algae consumption). This MoE value indicates low risk.

The percentage contribution to Hg intake from the consumption of 4 g/day is 1.73% of the TWI of organic Hg (1.3 μg/kg bw/week for methylmercury) (Table 6) [18]. Therefore, this does not pose a risk to the health of adults. According to the NOAEL of 0.04 mg/kg bw/day of methylmercury [18], the MoE for the consumption of arame algae is 182.

3.2. Nitrate Concentrations (NO₃⁻) and Dietary Intake

Table 7. Concentration of nitrates (mg/kg), standard deviations (SD), estimated daily intake, and contribution to the ADI of the analyzed algae.

Species	[NO3−] ± SD (mg/kg)	Max. Value	Min. Value	EDI (mg/day)	Contribution to ADI (%)
Arame	<500	-	-	-	-
Sea spaghetti	<500	-	-	-	-
Hijiki	<500	-	-	-	-
Kombu	3084 ± 2698	6400	<500	12.34	4.87
Sea lettuce	965 ± 135			3.86	1.52
Mixed salad	1096 ± 203	1239	952	4.38	1.73
Starry moss	<500	-	-	-	-
Nori	3183 ± 2279	>8000	<500	12.73	5.02
Wakame	<500	-	-	-	-

ADI, acceptable daily intake.

shows the mean nitrate concentrations in the analyzed algae as well as the estimated daily intake values and their percentage contribution to the ADI value set by EFSA in 2017 [24,25].

The nori species has the highest concentration of nitrates, with a mean value of 3183 mg/kg and with larger samples >8000 mg/kg. On the other hand, kombu seaweed has a mean concentration of 3084 mg/kg with a maximum value of 6400 mg/kg.

It should be noted that there is no legislation regulating the content of nitrates in seaweed. However, Commission Regulation (EU) No. 1258/2011 of 2 December 2011, which modified Regulation (EC) No. 1881/2006 with regard to the maximum content of nitrates in food products, set a limit of between 6000 and 7000 mg/kg for rocket or arugula [39]. Considering the limit applicable to arugula, whose permitted concentrations of nitrates are the highest of all foods, some kombu samples would exceed this limit, and therefore would not be suitable for human consumption.

However, taking into account the manufacturer’s recommended consumption of 4 g of nori algae per day, the mean concentration of 3183 mg/kg would represent a percentage contribution to the ADI of nitrates (3.7 mg/kg bw/day) of 5.02%. Although this would not pose a risk to adult health, it should be borne in mind that the storage and the cooking process of algae, etc. can lead to the transformation of nitrates into nitrites, thus posing a risk to the consumers’ health.

Although the algae analyzed do not suppose a high intake, the importance of taking into account other nitrate sources such as water and other vegetable foods should be noted. A study carried out by Qasemi et al. [40] reported nitrate levels in water from wells and springs of Azadshahr (northeastern Iran) of 1 up to 51 mg/L. Quijano et al. [41] determined the nitrate content in vegetables marketed in Valencia (Spain), finding concentrations of 40 mg/kg (carrots), 173.5 mg/kg (potatoes), and 1266.5 mg/kg (fresh spinach). It is advisable to moderate the consumption of the algae species that recorded the highest nitrate concentrations, especially in vegans or vegetarians, and by people living in areas of high concentrations of nitrate in the water supply.

4. Conclusions

Seaweed is becoming an increasingly important food in the diet of Western countries due to its nutritional profile. However, these organisms absorb and accumulate toxic metals such as Cd, Pb, and Hg or anions that, ingested in high concentrations, can trigger adverse effects, such as in the case of nitrates.

Asian algae have higher concentrations of Cd, Pb, and Hg than European algae. The Asian hijiki and nori algae are noteworthy for their Cd content. Some samples of nori algae presented NO₃⁻ concentrations above 8000 mg/kg. However, considering the consumption recommended by the manufacturers (4 mg/day), the maximum allowable intake values are not exceeded, nor are those set for toxic metals and nitrates. However, in the case of Cd, Pb, Hg, and NO₃⁻, legislation should be put in place to regulate their contents in edible algae, especially considering that the origin of the algae can affect their toxic metal and nitrate content. Monitoring studies should be conducted periodically to detect species or origins which could pose a risk from a toxicological point of view and to recommend avoiding their consumption. Similarly, the relevant authorities are urged to legislate for maximum levels of contaminants in these products to ensure their quality and food safety.