Comparison of Heavy Metals and Arsenic Species in Seaweeds Collected from Different Regions in Korea

Abstract

We evaluated the levels of heavy metals and arsenic (As) species in 11 different types of seaweed collected from major coastal cities in Korea. The concentration ranges of heavy metals in the seaweed were as follows: cadmium (0.023–0.232 mg/kg fresh weight [fw]), and lead (0.025–0.222 mg/kg fw), with most meeting international regulations for edible seaweeds. The amount of total As, however, was high, ranging from 1.020 to 20.525 mg/kg fw. Especially in the case of Sargassum seaweed, the fraction of inorganic As, including arsenate (As [V]) and arsenate (As [III]), which have potent toxicity, ranged from 5.198 to 16.867 mg/kg fw, while other seaweeds, such as Pyropia sp., Enteromorpha sp., Undaria sp., and Saccharina sp., predominantly contained a non-toxic organic As (i.d. arsenosugars). Multivariate analysis revealed that the Sargassum genus group had high levels of inorganic As. Sargassum seaweeds had a high fraction of inorganic As, but most of them are considered inedible seaweeds. Of these, Sargassum fusiforme (hijiki) is widely recognized as an edible seaweed, but the average daily intake is quite low based on statistical data from Asian countries and S. fusiforme is considered a safe food when eaten at the recommended daily intake.

1. Introduction

As one of the most nutritionally dense foods in a wide variety of vegetables, seaweed is particularly known for containing large amounts of bioavailable minerals that promote brain function, metabolism, and health, as well as vitamins [1,2,3,4], specifically vitamin K, which has a preventive role against cardiovascular disease and osteoporosis in postmenopausal women [5,6]. Seaweed is an abundant source of several phytochemicals, including the polysaccharide fucoidan, which has antiviral activity and can induce cancer cell death, and is spotlighted as a dietetic food due to its effects on decreasing the rate of sugar absorption [3,7,8]. Around the world, seaweed is also considered a ‘Super Food’ [9].

Like fish and shellfish, seaweed also has a propensity to absorb or store whatever is in the marine environment. Seaweed obtained from seawater contaminated by various substances, such as cadmium (Cd), lead (Pb), arsenic (As), or other heavy metals, will show accumulation of these contaminants [10]. Therefore, seaweed is frequently utilized as a bio-indicator of natural and anthropogenic contaminants for environmental monitoring to assess the health and sustainability of marine ecosystems [11,12,13,14]. Seaweed exposed to an environment contaminated by several toxic heavy metals must be thoroughly evaluated for its potential risk to human health through consumption [15].

Approximately 28 million tons of seaweed are produced and cultivated each year in almost every country, and over 95% of the total output is produced by Asia [16]. Seaweed has been a prominent component of Asian diets and medicines from the distant past to the present, particularly in East Asia, such as in Korea, Japan, and China. The total output of seaweed in Korea, the fourth largest producer of seaweed in the world, is approximately 1.2 million tons [16], and the area of seaweed production is distributed widely throughout the entire sea of Korea. Brown (66% of total output) and red (32% of total output) seaweeds are most common, and major species include Saccharina japonica (sea tangle), Undaria pinnatifida (sea mustard), and Pyropia tenera (laver) [16].

The average daily intake of seaweed in Korea is about 23.8 g, which is significantly higher than in other countries. Therefore, the seaweeds containing heavy metals that are growing in coastal seawater may have unknowingly harmed Koreans [17]. According to research in 2021, exposure to the heavy metals Cd, Pb, and As in foods was 0.358, 0.192, and 3.454 µg/kg/day in Korea, respectively. Surprisingly, when As exposure through each kind of seafood was converted to the 95th percentile confidence limit, seaweed accounted for most of these heavy metal contaminants [18]. Concerns regarding the harmful effects of chronic As exposure due to seaweed ingestion are widely known, but the data regarding food safety of heavy metals, including As, by type of seaweed and edibleness of each type of seaweed is insufficient. In particular, the family Sargassaceae, in contrast to other seaweeds, contains considerable amounts of inorganic As, such as arsenate (As[V]) and arsenite (As[III]), which are extremely toxic under both acute and chronic exposures [19].

Additionally, although recent studies have evaluated the accumulation levels and health risks of various heavy metals in marine organisms, global emphasis has been on studies in fish or shellfish, with seaweed rarely being assessed. A few studies have found high concentrations of heavy metals in the blood and significantly higher concentrations of As in the urine of Asians who consistently consumed a moderate amount of seaweed compared with low or non-consumers [20,21,22,23]. These results indicate that a large amount of intake of seaweed may be extremely harmful and lead to various diseases. Therefore, with respect to the possibility of toxic intake in food, research related to setting appropriate intake standards, such as monitoring and risk assessment of exposure to As species and heavy metals such as Cd, aluminum, and Pb in seaweeds, is gradually increasing.

The present study investigated the major heavy metal contents in several species of seaweed and evaluated the potential health risks of Cd, Pb, and As through the consumption of seaweed harvested from sea areas in Republic of Korea (ROK). Additionally, six As species contained in seaweeds were precisely analyzed to confirm the major As species in seaweeds and compared with those in other marine organisms. Statistical analysis was performed to determine a systematic correlation among the measured values of each component of heavy metals in seaweeds. These results are very valuable for understanding the risk of consuming seaweed and the contamination of seaweed by heavy metals.

2. Materials and Methods

2.1. Reagents and Standard Solutions

All reagents used were 98% pure or higher grade reagents, and deionized water (DIW) was passed through an arium^® water purification system (Satorious, Göttigen, Germany). Supra pure grade nitric acid (Merck, Darmstadt, Germany) and HPLC grade methanol (Merck) were used for preparation of the analysis sample solution. For the analysis of Cd, Pb, and total As, a series of calibration standards was prepared from multi-element calibration standards (Perkin Elmer, Waltham, MA, USA). Arsenocholine (AsC; Wako, Kyoto, Japan), arsenobetaine (AsB; Wako), monomethylarsonic acid (MMA; Millipore Sigma, St. Louis, MO, USA), dimethylarsinic acid (DMA; Millipore Sigma, Burlington, MA, USA), arsenite (As[III]; SPEX CertiPrep, Metuchen, NJ, USA), and arsenate (As[V]; SPEX CertiPrep) were used to prepare the As species standard solutions. In addition, the HPLC mobile phase for the analysis of the Arsenic (As) species was prepared by adding sodium 1-butane sulfate, malonic acid, and tetramethylammonium hydroxide (Millipore Sigma).

2.2. Sample Collection

In 2019, seaweed samples were collected from joint fishery product auction houses (Donghae: Mukho harbor fisheries market; Samchuk: Samcheok fisheries market; Uljin: Jukbyun fisheries market; Youngduk: Donggwang fisheries market; Pohang: Jukdo market; Gyeongju: Yangbuk fisheries market; Ulsan: Agricultural and marine products market; Busan: Jagalchi market; Geoje: Geoje fisheries market; Tongyoung: Chungang market; Yeosu: Yeosu fisheries market; Boseong: Boseong fisheries market; Wando: Wando suhyup fisheries market; Haenam: Maeil market; Jeju: Dongmun fisheries market; Boryoung: Muchangpo fisheries market; Taean: Anmyeondo fisheries market) selling various seaweeds and marine animals in representative coastal cities of ROK, in consideration of the regional production of seaweed (Figure 1). Identification of the seafood samples was confirmed on the basis of the advice of related experts. A total of 37 species were used for analysis of heavy metals and As species in this study. Eleven species of seaweed, including laver (Pyropia sp.), green laver (Enteromorpha sp.), sea mustard (Undaria pinnatifida), sea tangle (Saccharina japonica), and gulf-weed or hijiki (Sargassum spp.), were collected. In addition, for comparative analysis of levels of As species in seaweeds and those in marine animals, 3 species of crustaceans (Portunus trituberculatus, Solenocera melantho, Chionoecetes opilio), 4 species of cephalopods (Octopus ocellatus, Todaroldes pacificus, Octopus minor, Enteroctopus dofleini), 13 species of fish (Larimichthys polyactis, Sebastes schlegelii, Lateolabrax jaonicus, Gadus macrocephalus, Seriola quinqueradiata, Chelon haematocheilus, Pagrus major, Scomber japonicus, Trichiurus lepturus, Miichthys miiuy, Doederleinia berycoides, Thunnus thynnus, Cololabis saira), and 6 species of shellfish (Ruditapes philippinarum, Crassostrea gigas, Meretrix petechialis, Argopecten irradians, Mytilus galloprovincialis, Atrina pectinata) were collected during the same period. All samples were directly transported to the laboratory in coolers at temperatures below 10 °C.

2.3. Sample Preparation

Upon arrival at the laboratory, the collected samples were immediately separated according to species, washed and rinse washed with DIW to remove surface impurities. The seaweed samples were lyophilized using a vacuum freeze dryer (FDU-2100, EYELA, Tokyo, Japan) and finely powdered in a mixer. The edible tissues of the marine animal samples were prepared by removing the inedible parts. The tissue samples were homogenized and weighed. The homogenized tissues were freeze-dried with a freeze dryer, and then ground into powder for analysis. All powder samples were analyzed for Cd, Pb, total As, and As species.

2.4. Analysis of Cd, Pb, and Total As

To analyze Cd, Pb, and total As, 1.0 g powdered sample was placed in a 20 mL digestion vessel, to which 5 mL nitric acid was added. The samples were digested using a high-pressure sample decomposition system (UltraWAVE, Mileston, Sorisole, Italy) and analyzed for heavy metals. The digested samples were allowed to cool to room temperature, dissolved in 2% nitric acid, filtered, and diluted to 100 mL of 2% nitric acid for analysis of heavy metals.

All digested samples were analyzed in triplicate for Cd, Pb, and total As using an inductively coupled plasma mass spectrometer (Nexion 300D, Perkin-Elmer, Waltham, MA, USA).

2.5. Analysis of As Species

To analyze As species, 0.5–1.0 g of sample was weighed in a screw-top centrifuge tube and extracted with 10 mL of 50% (v/v) methanol in 1% HNO₃. Extraction was performed using an ultrasonic bath for 1 h. After centrifuging at 6000× g for 10 min, the supernatant was transferred into a 50 mL polyethylene tube, and the extraction step was repeated twice. The supernatant extracts were filtered and kept at 4 °C until analysis.

The As species were separated using Capcell Pak C18MG (4.6 mm × 250 mm × 5 μm; Shiseido Co., Ltd., Tokyo, Japan) and individual concentrations of As species were determined using an inductively coupled plasma mass spectrometer connected in line to a liquid chromatographer (Flexar FX-10, Perkin-Elmer, Waltham, MA, USA). The chromatographic conditions employed were a modification of Hirata’s method [24]. The mobile phase was a 4 mM sodium 1-butane sulfate, 4 mM malonic acid, and 10 mM tetramethylammonium hydroxide (pH 2.3). The quadrupole mass analyzer was operated in the single ion monitoring mode (m/z 91) for detecting As and dynamic reaction cell (DRC) mode using oxygen (

Table 1. LC-ICP/MS operating condition and parameters for As speciation.

LC Conditions
Column	Capcell Pak C18MG (4.6 mm × 250 mm × 5 μm)
Injection volume	20 μL
Mobile phase	4 mM sodium 1-butane sulfate, 4 mM malonic acid, and 10 mM tetramethylammonium hydroxide (pH 2.7 or lower)
Run time	15 min
Pump steps	Equilibration for 0.5 min; 1.2 mL/min, Run for 15 min; 0.75 mL/min
Oven temperature	20 °C
ICP-MS parameters
Rf power	1600 W
Deflector voltage (eV)	−5 eV
Ar plasma gas flow rate	18 mL/min
Ar nebulizer gas flow rate	0.90 mL/min
Sample introduction	Concentric nebulizer
Dwell time	250 ms
Analyze mode	AsO 91 (90.9165 amu)/DRC Mode
DRC conditions	O2 gas flow: 0.5 mL/min, Rpq: 0.45

). The blanks, calibration standards, and CRMs for testing the As species were also analyzed using the same methods. Unidentified As species were quantified using a calibration curve created based on As(V). The levels of As species are expressed in mg/kg fresh weight (fw).

2.6. Quality Assurance

The analysis methods followed were validated by measuring several quality parameters, including linearity, limit of detection (LOD), limit of quantification (LOQ), recovery, accuracy and precision [25,26]. LOD and LOQ were calculated with ten times standard deviation in seven replicates of standard solutions. Linearity was evaluated from the calibration curve of heavy metals and As species standards with at least six different concentrations, including the blank. The recovery, precision (%C.V), and accuracy (%bias) of the analytical method for seaweed were tested by analyzing the CRM 7405a (Hijiki powder; National Institute of Advanced Industrial Science and Technology, Japan) and SRM 3232 (Kelp power; National Institute of Standard and Technology, USA) for the determination of Cd, Pb, As, and As(V). For fish and shellfish, the CRM DORM-4 (Fish protein; National Research Council, Canada) and SRM 1566b (Oyster issue; National Institute of Standard and Technology, USA) were also analyzed for quality assurance of Cd, Pb, and As determination.

2.7. Statistical Analysis

Principal component analysis (PCA) of multivariate statistical analysis was performed in the R programming environment (http://cran.r-project.org, accessed on 13 December 2019) using the VEGAN [27] package at a 95% confidence level to investigate the correlation between heavy metals (Pb, Cd, As), six As species, and marine organisms including seaweed.

3. Results and Discussion

3.1. Validation of Analytical Method

The value of LOD and LOQ were in the range of 0.114–0.185 μg/kg and 0.363–0.590 μg/kg for heavy metals, 0.064–0.108 μg/kg and 0.213–0.359 μg/kg for As species, and the values of correction coefficient (R²) calculated for all calibration curves were in the range of 0.9992–0.9997 for heavy metals, and 0.9996–0.9997 for As species (

Table 2. Quality parameters of analytical methods for determination of heavy metals and As species.

Parameters	Heavy Metals			As Species
	Cd	Pb	As	As(V)	As(III)	MMA	DMA	AsB	AsC
Linearity (R2)	0.9992	0.9996	0.9997	0.9998	0.9999	0.9999	0.9998	0.9996	0.9998
LOD (μg/kg) a	0.114	0.126	0.185	0.067	0.098	0.108	0.080	0.064	0.080
LOQ (μg/kg) b	0.363	0.402	0.590	0.223	0.328	0.359	0.268	0.213	0.268

^a LOD = 3.143 × σ (σ standard deviation among seven replicates in the determination of standard solution). ^b LOQ = 10 × σ (σ standard deviation among seven replicates in the determination of standard solution).

). The LC-ICP/MS chromatogram for standard solution of As species is presented in Figure 2. In this analytical method for As species, chromatographic peak separation was investigated. In

Table 3. Quality parameters of analytical methods for the determination of heavy metals and As species by sample matrix.

Analyte	Certified Value (mg/kg)	Observed Value (mg/kg)	Recovery (%)	Accuracy (%) a	Precision (%) b
CRM 7405-a
Cd	0.79 ± 0.02	0.77 ± 0.05	97.5	−2.53	5.93
Pb	0.43 ± 0.03	0.42 ± 0.03	96.9	−3.06	7.89
As	35.8 ± 0.9	32.7 ± 2.7	91.3	−8.71	8.27
As(V)	10.1 ± 0.5	9.7 ± 0.2	95.6	−4.41	1.85
SRM-3232
Cd	0.426 ± 0.008	0.397 ± 0.025	93.2	−6.81	6.21
Pb	1.032 ± 0.039	1.087 ± 0.066	105.4	5.36	6.05
As	38.3 ± 1.3	34.9 ± 2.8	91.0	−8.99	8.16
DMA	0.479 ± 0.077	0.409 ± 0.019	85.4	−14.64	4.6
SRM-1566b
Cd	2.48 ± 0.08	2.35 ± 0.18	94.9	−5.11	7.67
Pb	0.308 ± 0.009	0.301 ± 0.017	97.6	−2.37	5.67
As	7.65 ± 0.65	7.56 ± 0.56	98.8	−1.18	7.42
CRM-DORM-4
Cd	0.306 ± 0.015	0.284 ± 0.025	92.8	−7.24	8.88
Pb	0.416 ± 0.053	0.372 ± 0.011	89.3	−10.68	2.85
As	6.80 ± 0.64	5.93 ± 0.09	87.2	−12.80	1.63

^a Accuracy (%Bias) = (mean value of seven replicates for determination−certified concentrations )/(certified concentrations) × 100%; ^b precision (%C.V) = (standard deviation among seven replicates for determination)/(mean value of seven replicates for determination) × 100%.

, for CRMs analyzed using the sample matrix, the %bias values and %C.V were below ±15% and below 10% deviation from the certified values. In addition, the recovery percentages were in the range of 85.4–105.4% for heavy metals and 95.6% for inorganic As, which is within the acceptable criteria [26,28]. Moreover, extraction efficiencies (%mass balance; sum of As species/total As × 100) were in the range of 72.7–95.4% for seaweeds, 107.8–110.6% for crustaceans, 93.6–116.7% for cephalopods, 63.6–121.7% for fish, and 61.8–86.3% for shellfish, respectively (

Table 4. Concentrations of heavy metals and As species in seaweed, crustacean, and cephalopods (mg/kg, fw).

Scientific Name	Cd	Pb	As	As(Ⅴ)	As(III)	MMA	DMA	AsB	AsC	Unidentified As Species	Sum of As Species	Extraction Efficiency (%)
Seaweed
Pyropia spp.	0.043 ± 0.042	0.116 ± 0.161	1.226 ± 0.787	0.015 ± 0.010	<DL a	<DL	<DL	0.015 ± 0.003	<DL	0.889 ± 0.049	0.919 ± 0.036	74.9
Enteromorpha spp.	0.038 ± 0.002	0.179 ± 0.012	1.020 ± 0.100	0.010 ± 0.005	<DL	0.011 ± 0.015	0.015 ± 0.000	0.021 ± 0.001	<DL	0.685 ± 0.119	0.742 ± 0.097	72.7
U. pinnatifida	0.085 ± 0.084	0.051 ± 0.039	4.062 ± 0.977	0.015 ± 0.005	0.003 ± 0.005	0.017 ± 0.018	<DL	0.694 ± 0.444	<DL	2.481 ± 0.129	3.210 ± 0.563	79.0
S. japonica	0.023 ± 0.002	0.025 ± 0.019	8.616 ± 0.545	0.035 ± 0.036	<DL	0.011 ± 0.010	0.004 ± 0.007	3.026 ± 0.920	0.157 ± 0.209	3.880 ± 0.807	7.114 ± 1.513	82.6
S. fusiforme	0.071 ± 0.099	0.044 ± 0.030	12.158 ± 3.245	9.639 ± 4.355	0.214 ± 0.123	0.004 ± 0.008	0.032 ± 0.027	0.201 ± 0.056	0.329 ± 0.073	0.796 ± 0.435	11.214 ± 4.786	92.2
S. thunbergii	0.074 ± 0.020	0.087 ± 0.039	15.430 ± 7.869	12.882 ± 6.737	0.283 ± 0.141	<DL	0.022 ± 0.012	0.381 ± 0.119	0.180 ± 0.237	0.974 ± 0.116	14.723 ± 7.323	95.4
S. miyabei	0.154 ± 0.018	0.222 ± 0.258	8.093 ± 3.707	5.012 ± 3.074	0.186 ± 0.056	<DL	0.014 ± 0.008	0.216 ± 0.207	0.098 ± 0.111	0.791 ± 0.448	6.318 ± 3.415	78.1
S. confusum	0.220 ± 0.008	0.077 ± 0.056	13.169 ± 1.292	10.867 ± 0.967	0.189 ± 0.139	<DL	0.006 ± 0.008	0.115 ± 0.033	0.199 ± 0.113	0.945 ± 0.422	12.321 ± 1.681	93.6
S. yezoense	0.232 ± 0.276	0.155 ± 0.170	20.524 ± 1.562	16.697 ± 2.017	0.170 ± 0.007	0.055 ± 0.078	0.006 ± 0.009	0.507 ± 0.173	<DL	1.207 ± 0.306	18.642 ± 1.463	90.8
S. hemiphyllum	0.191	0.062	19.383	14.356	0.251	0.016	0.014	0.458	<DL	1.601	16.696	86.1
S. micracanthum	0.046	0.046	13.686	9.378	0.193	0.029	0.059	0.119	<DL	1.021	10.798	78.9
Crustaceans
P. trituberculatus	0.102 ± 0.082	0.014 ± 0.013	9.049 ± 0.409	<DL	<DL	0.012 ± 0.017	<DL	9.366 ± 2.334	0.077 ± 0.109	0.148 ± 0.026	9.603 ± 2.452	106.1
S. melantho	0.028 ± 0.007	0.016 ± 0.009	14.841 ± 0.302	<DL	<DL	0.040 ± 0.056	<DL	15.727 ± 1.664	<DL	0.233 ± 0.046	16.000 ± 1.654	107.8
C. opilio	0.045 ± 0.029	0.043 ± 0.028	28.491 ± 12.850	<DL	<DL	0.046 ± 0.014	<DL	29.407 ± 11.459	0.042 ± 0.060	2.017 ± 1.533	31.512 ± 13.067	110.6
Cephalopods
O. ocellatus	0.115 ± 0.038	0.042 ± 0.005	5.064 ± 0.403	<DL	<DL	0.004 ± 0.006	<DL	4.629 ± 0.057	0.113 ± 0.016	0.050 ± 0.029	4.796 ± 0.050	94.7
T. pacificus	0.331 ± 0.222	0.022 ± 0.000	6.747 ± 0.762	<DL	<DL	0.003 ± 0.004	<DL	6.273 ± 0.758	0.010 ± 0.014	0.031 ± 0.004	6.317 ± 0.736	93.6
O. minor	0.038 ± 0.043	0.107 ± 0.022	21.229 ± 12.374	<DL	<DL	0.015 ± 0.013	0.011 ± 0.010	24.387 ± 14.916	0.025 ± 0.030	0.334 ± 0.182	24.772 ± 15.122	116.7
E. dofleini	0.108 ± 0.041	0.053 ± 0.035	34.037 ± 5.083	<DL	<DL	0.007 ± 0.010	0.030 ± 0.013	38.013 ± 6.883	<DL	0.509 ± 0.277	38.559 ± 6.609	113.3

^a <DL = below detection limit.

and

Table 5. Concentrations of heavy metals and As species in fish and shellfish (mg/kg, fw).

Scientific Name	Cd	Pb	As	As(Ⅴ)	As(III)	MMA	DMA	AsB	AsC	Unidentified As Species	Sum of As Species	Extraction Efficiency (%)
Fish
L. polyactis	0.002 ± 0.000	<DL	2.173 ± 0.220	0.006 ± 0.001	<DL	0.006 ± 0.008	0.019 ± 0.027	2.436 ± 0.053	<DL	0.033 ± 0.011	2.499 ± 0.081	115.0
S. schlegelii	0.001 ± 0.000	0.002 ± 0.003	3.076 ± 0.729	0.005 ± 0.000	<DL	<DL	0.002 ± 0.002	3.409 ± 0.946	<DL	0.089 ± 0.059	3.504 ± 1.002	113.9
L. jaonicus	0.001 ± 0.000	0.004 ± 0.004	1.425 ± 0.020	<DL	<DL	0.016 ± 0.008	0.021 ± 0.010	1.688 ± 0.148	<DL	0.007 ± 0.003	1.732 ± 0.127	121.5
G. macrocephalus	0.002 ± 0.000	0.002 ± 0.002	8.356 ± 3.104	0.002 ± 0.002	0.003 ± 0.004	0.001 ± 0.002	0.007 ± 0.003	7.906 ± 1.216	0.020 ± 0.028	0.118 ± 0.045	8.055 ± 1.146	96.4
S. quinqueradiata	<DL a	0.001 ± 0.001	1.871 ± 0.026	<DL	0.009 ± 0.012	0.027 ± 0.010	0.031 ± 0.018	2.023 ± 0.024	0.017 ± 0.025	0.022 ± 0.021	2.130 ± 0.073	113.8
C. haematocheilus	<DL	0.002 ± 0.003	0.817 ± 0.091	0.002 ± 0.002	<DL	0.006 ± 0.009	0.016 ± 0.006	0.493 ± 0.132	<DL	0.003 ± 0.004	0.520 ± 0.127	63.6
P. major	<DL	0.001 ± 0.000	3.025 ± 3.132	<DL	<DL	0.013 ± 0.018	0.004 ± 0.005	2.076 ± 2.090	<DL	0.917 ± 0.039	3.010 ± 2.063	99.5
S. japonicus	0.001 ± 0.000	<DL	1.987 ± 1.127	<DL	0.003 ± 0.004	0.022 ± 0.023	0.051 ± 0.030	1.695 ± 0.628	<DL	0.035 ± 0.034	1.805 ± 0.665	90.8
T. lepturus	0.001 ± 0.000	<DL	1.463 ± 0.303	0.011 ± 0.007	0.002 ± 0.003	0.003 ± 0.004	0.017 ± 0.024	1.697 ± 0.134	<DL	0.051 ± 0.010	1.781 ± 0.114	121.7
M. miiuy	<DL	<DL	0.468 ± 0.051	0.006 ± 0.009	<DL	<DL	<DL	0.511 ± 0.069	<DL	0.011 ± 0.016	0.528 ± 0.063	112.9
D. berycoides	0.004 ± 0.005	<DL	2.231 ± 0.800	<DL	0.009 ± 0.012	0.014 ± 0.008	0.002 ± 0.003	2.088 ± 0.418	<DL	0.148 ± 0.014	2.261 ± 0.430	101.3
T. thynnus	0.001 ± 0.001	<DL	1.617 ± 0.820	<DL	<DL	<DL	0.050 ± 0.018	1.691 ± 1.259	<DL	0.031 ± 0.004	1.772 ± 1.281	109.6
C. saira	0.012 ± 0.001	0.011 ± 0.004	0.753 ± 0.028	<DL	<DL	0.047 ± 0.005	0.062 ± 0.032	0.458 ± 0.060	<DL	0.069 ± 0.097	0.636 ± 0.074	84.4
Shellfish
R. philippinarum	0.150 ± 0.054	0.211 ± 0.042	7.137 ± 1.750	0.007 ± 0.010	0.009 ± 0.003	0.059 ± 0.025	0.050 ± 0.012	3.928 ± 2.132	0.321 ± 0.024	0.266 ± 0.084	4.639 ± 2.243	65.0
C. gigas	0.615 ± 0.014	0.144 ± 0.064	3.609 ± 1.487	<DL	0.002 ± 0.003	0.036 ± 0.013	0.108 ± 0.020	1.542 ± 0.371	0.194 ± 0.122	0.451 ± 0.046	2.333 ± 0.298	64.6
M. petechialis	0.155 ± 0.003	0.045 ± 0.015	3.249 ± 0.115	<DL	0.004 ± 0.006	<DL	0.011 ± 0.016	1.326 ± 0.021	0.257 ± 0.022	0.616 ± 0.232	2.214 ± 0.178	68.2
A. irradians	2.447 ± 1.714	0.363 ± 0.254	1.955 ± 0.145	0.007 ± 0.010	<DL	<DL	0.048 ± 0.040	0.997 ± 0.059	0.020 ± 0.007	0.281 ± 0.157	1.353 ± 0.121	69.2
M. galloprovincialis	0.518 ± 0.410	0.143 ± 0.020	3.671 ± 2.252	0.008 ± 0.011	0.001 ± 0.002	0.020 ± 0.021	0.048 ± 0.013	1.385 ± 0.867	0.221 ± 0.236	0.587 ± 0.387	2.270 ± 1.481	61.8
A. pectinata	0.155 ± 0.014	0.022 ± 0.004	3.867 ± 0.200	<DL	<DL	<DL	<DL	3.124 ± 0.128	0.170 ± 0.042	0.044 ± 0.017	3.338 ± 0.103	86.3

^a <DL = below detection limit.

). Similarly, Hirata [24] reported that extraction efficiencies were ranged from 52.9 to 112.3% in marine samples. Based on these results for quality assurance, the analytical methods followed were found to be very efficient for application in the determination of heavy metal and As species in seaweeds and marine animals.

3.2. Concentrations of Heavy Metals and Arsenicals in Seaweeds

In general, most seaweeds adsorb and accumulate minerals and trace elements from the environment they inhabit, and effectively accumulate heavy metals like Cd, Pb, and As. The concentrations of heavy metals are summarized in Table 4. The concentrations of total As were mostly dominant in all of the collected seaweeds (Pyropia, Enteromorpha, Undaria, Saccharina, and Sargassum), whereas the concentrations of the other contaminants were marginal (<1.0 mg/kg).

The Cd concentrations of the seaweeds ranged from 0.023 to 0.232 mg/kg fw. Sargassum spp. had slightly higher Cd concentrations than the other seaweeds. Among the brown algae variety of seaweeds, Sargassum spp. is considered an effective biosorbent of heavy metals, including Cd, from contaminated effluents [29,30,31]. Hashim and Chu [32] compared the biosorption behavior of various brown, green, and red seaweeds and reported that the brown seaweed Sargassum spp. had excellent Cd absorption capabilities. In the present study, however, the Cd concentration in Sargassum varied widely, from 0.046 to 0.232 mg/kg fw. U. pinnatifida is also considered a suitable adsorbent for heavy metal pollutants [33,34], but the Cd concentration of U. pinnatifida, including the other seaweeds, was below 0.1 mg/kg.

The concentrations of Pb in the different seaweeds ranged from 0.025 to 0.222 mg/kg, and all samples were suitable for consumption under Chinese regulations (Pb < 1 mg/kg in seaweeds). These results were similar to those reported in other studies (<0.3 mg/kg fw) in ROK [35].

As mentioned above, the results of variability in heavy metal concentrations in seaweeds may be due to differences in bioabsorption, due to factors such as seasonal or local variations, algal growth conditions, environmental factors (temperature, salinity, light, and pH), and the presence of industrial complexes or cities nearby [36,37].

The concentrations of total As in the examined seaweeds ranged from 1.020 to 20.524 mg/kg fw, and were generally higher compared with the other heavy metals. Total As concentrations are generally higher in marine organisms than in terrestrial organisms [38], and accordingly, seafood such as fish, shellfish, and seaweeds exhibit much higher total As contents when compared to other kinds of foods.

A considerable portion of As ingested by humans originates from the variety of seafood included in their diet [39]. The As compounds are classified as largely belonging to two forms, organic and inorganic species, which differ greatly with respect to their toxicity [40]. Organic As compounds mainly comprise MMA, DMA, AsC, and AsB. Of these, MMA and DMA were rarely found in seaweeds, as shown in Table 4, while DMA was detected almost exclusively in fish viscera (such as stomach, intestines, liver, and gills) [41]. AsC in seaweeds was also detected in low concentrations because it is rapidly transformed into AsB through the process of As metabolism [42,43]. In other words, AsC plays a minor role as a metabolic precursor of AsB in the marine ecosystem [44].

As shown in Table 4 and Table 5, however, the As species in the tissues or muscles of crustaceans, cephalopods, fish, and shellfish, which are mainly consumed by humans, mostly present in the form of AsB, produced by the endpoint of the As cycle in the marine environment, depending on specific microbial organisms [45], which is known to be a non-toxic and easily excreted material. Inorganic As species, particularly arsenite As(III) and arsenate As(V), which are much more toxic than organic species, comprise a small proportion of the total As in seafood. In this study, a relatively low amount of inorganic As species compared with the total amount of As was shown in crustaceans, cephalopods, fish, and shellfish (Table 4 and Table 5). Similarly, Donohue and Abernathy [46] reported that As(III) and As(V) account for less than 3% of the total amounts of As in fish and crustaceans. Therefore, in most countries, the standard regulatory As intake limit is determined on the basis of inorganic As levels rather than total As amounts, due to these differences in toxicity.

Seaweeds also contain high levels of arsenicals, and the concentrations of total As are generally higher in brown algae than in green and red algae (Table 4) [38]. Most are bound in organic forms, which are not acutely toxic like the inorganic species, but interestingly, Sargassum spp. in brown algae had the highest amount of arsenate As(V) among six arsenicals. The Sargassum spp. group contained high levels of inorganic As (summation of As[V] and As[III]), accounting for approximately 69% to 85% of the total As concentration, but others can be seen to have less than 1.5%. On the other hand, other groups of seaweeds contained high levels of unidentified As (approximately 45% to 72%) (Table 4). Narukawa [47] reported that the main As species in seaweed, excluding the Sargassum spp. group, were asenosugars. Despite the fact that, because of the absence of asenosugar group standards, the unidentified species is unclear, it is likely an arsenosugar. Therefore, these results show that the Sargassum spp. group contains high concentrations of inorganic As when compared to other seaweeds. Narukawa [47] reported that the major As species in seaweed belonged to the arsenosugar group, except that in the case of Sargassum spp., the asenosugar group was not certain as there were no standards; therefore, the unidentified species are likely to belong to the group of arsenosugars. This is because Sargassum spp. has a higher mineral content than other seaweeds.

Reasons for the large amount of inorganic As in most Sargassum seaweeds have been proposed in several related studies. Rose et al. [48] demonstrated that two main reasons were (i) lack of metabolic conversion ability from inorganic forms to organic forms, caused by the efficient use of energy or genetic differences; and (ii) the accumulation of inorganic As as a specific defense mechanism against predation. In addition, oxy-anions such as As(V) and As(III), which are taken up in the marine environment efficiently induce the biosynthesis of phytochelatins, which helps store inorganic As in Sargassum seaweeds [49,50].

When the concentrations of As compounds in seaweed obtained from this study were compared with those in previous studies, most of the As values demonstrated a tendency consistent with that of inorganic As being relatively higher in Sargassum spp., including Hijiki, than in other seaweeds [51,52,53].

Although the levels of arsenicals in various seaweeds from different countries were compared with those in this study, it is difficult to thoroughly compare data from several authors due to differences in sample treatments and analytical methods. Therefore, it is important to establish international best practices for analyzing arsenicals as soon as possible.

3.3. Multivariate Analysis

For best results, all species to unit variance was standardized using the package VEGAN in the R package for showing a more balanced ordination plot, because only abundant specific As species such as As(V) and AsB with high variances were sufficiently explained. Additionally, the points or labels for marine organisms and As species were represented using the standard ordination plot command, and biplot arrows for six arsenicals and clustering groups in PCA were used to elucidate the correlation between As and seaweeds or marine animals.

The correlation results obtained from the PCA revealed associations between six As compounds (As[V], As[III], MMA, DMA, AsB, and AsC) and seaweeds with different types of marine organisms (Figure 3), while the multivariate analysis was performed in many ways such that there was no direct relationship between them and heavy metals (Cd, Pb, and As). Six groups classified on the basis of the levels of the six arsenicals were additionally confirmed by biplot of PCA: Group A, associated with Sargassum spp. in brown seaweeds (with exclusively higher inorganic As than others); Group B, associated with Pyropia sp., Enteromorpha sp., Undaria sp., and Saccharina sp. in the other seaweeds (with no detection or low values for six As compounds); Group C, associated with crustaceans (with high AsB than others); Group D, associated with cephalopod (with relatively higher AsB than others); Group E, associated with fish (with relatively higher AsB than others); Group F, associated with shellfish (with high MMA and DMA). In this analysis, we checked again that inorganic As had a decisive effect on clustering only Sargassum seaweeds and this group was a food safety threat because of highly toxic inorganic As. Yokoi and Konomi [54] suggested that the variations in the levels of inorganic As were due to specific seaweeds like the family Sargassaceae rather than environmental factors or the origin of the samples.

Principal component 1 (PC1; 36.20% of the variance) was clearly associated with major inorganic As and organic As, where, when divided into two types, the group comprising Sargassum spp. contained As(V) and As(III) and the other groups contained AsB. PC2 (23.25% of the variance) was slightly associated with minor components such as MMA, DMA, and AsC, which differentiated seaweeds and cephalopods from other marine animals like crustaceans, fish, and shellfish.

3.4. Risk and Hazard Assessment in Seaweeds

Seaweeds are regularly consumed and are increasingly being utilized as a main component of the diet, both in ROK and other parts of Asia. Several kinds of seaweeds are consumed in greater quantities than others. For example, both dried and seasoned types of laver (Phyropia spp.) in red seaweed are highly popular as rice dishes, and up to 1 g dry weight might be consumed at every meal in ROK. Similarly, sea mustard (U. pinnatifida) and sea tangle (S. japonica) in brown seaweeds are used for making soups, seasoning materials, and salads; therefore, considerable quantities could be consumed. Other brown seaweeds, such as gulf-weed (Sargassum spp.), are occasionally used as side dishes mixed with a variety of vegetables. Of these, S. fusiforme (hijiki), an edible seaweed, is used to supplement various dishes and salads.

As shown in Table 4, the concentrations of Pb and Cd were at safe levels that are below the maximum threshold values recommended by France (Pb < 5 mg/kg dw and Cd < 0.5 mg/kg dw), China (Pb < 1 mg/kg dw) and ROK (Cd < 0.3 mg/kg dw for only laver). These results are considered to meet food safety standards. However, most Sargassum seaweeds can be classified as high-risk foods, because the inorganic As concentrations in them exceed the regulations and limits of several countries, including France, Spain, Australia–New Zealand, and China (3.0, 2.0, 1.0 and 0.3 mg/kg dw, respectively). Although only a few species, such as S. fusiforme and S. thunbergii, of Sargassum spp. are used as edible seaweeds, the levels of inorganic As in both S. fusiforme and S. thunbergii were 9.853 and 13.166 mg/kg fw, respectively, and thus their consumption in large amounts may be unsafe.

Many countries have actively regulated the intake and import of Sargassum seaweeds, particularly S. fusiforme. The Canadian Food Inspection Agency warned the public to abstain from eating S. fusiforme. In 2004, the European Commission and the UK Food Standards Agency issued a risk alert for S. fusiforme. Food Standards Australia–New Zealand and the Hong Kong Centre for Food Safety publicly warned against S. fusiforme and discontinued its import and sales. In Japan, S. fusiforme was publicized as a health food rich in all kinds of vitamins and minerals, but the governmental guidelines state that intake of S. fusiforme should be restricted to less than 4.7 g daily, and even lower for pregnant women, children, and those in poor health.

Contrary to popular concerns, the Sargassum group of seaweed is rarely consumed. Among them, S. thunbergii is utilized as a material for stock feed or medicines (antihelminthic components) rather than food [55], and while S. fusiforme is a typical edible seaweed in this group, the mean daily intake is very low, ~0.11 g, according to the statistical standards of ROK [56]. Moreover, most of As in the edible Sargassum group, including in S. fusiforme, is removed during the production and cooking processes [57]. The Joint FAO/WHO Expert Committee on Food Additives (JECFA) noted that the provisional tolerable weekly intake of 15 μg/kg body weight (bw; equivalent to 2.1 µg/kg bw/day) was established in 1989. The provisional tolerable weekly intake based on the concentration of inorganic As for S. fusiforme obtained from the present study was 0.078–0.179 µg/kg bw, and it was confirmed that these values meet the standard proposed by the JECFA. In 2015, the European Commission reported that the current standard of inorganic As for weekly intake was no longer appropriate due to adverse effects found at exposure levels that are lower than the JECFA proposed. Therefore, it was redefined using the benchmark dose (cancers of the lung, skin, and bladder, as well as skin lesions) to the confidence level, which is (BMDL01) between 0.3 and 8 μg/kg bw per day.

Therefore, upon further review, the threshold value of daily inorganic As intake through seaweeds (especially Sargassum spp.) suited to the eating habits of Koreans might be established by other evaluation methods, including the BMDL approach. In addition, when Sargassum species is used as a major source of stock feed, it should be evaluated for inorganic As toxicity for all types of farmed animal.

4. Conclusions

We compared the levels of heavy metals and major As species in seaweeds with those in other marine organisms. The concentrations of heavy metals such as Pb and Cd in most seaweeds were below the standard values recommended by international regulations and those of the Korea Food Code. The total As content in Sargassum spp. of brown seaweeds was relatively higher than that in other seaweeds, and they contained considerable amounts of inorganic As, As(V) in particular. Therefore, Sargassum seaweeds are classified as a high-risk food group around the world due to concerns related to the high toxicity of inorganic As. Fortunately, most Sargassum spp. are inedible, and although S. fusiforme (hijiki) is a widely known edible seaweed, the mean daily intake is quite low according to the statistical data on Korean and Japanese diets, where it is recognized that the ingestion of seaweeds is relatively higher than that in other countries. Therefore, if seaweeds such as Sargassum spp. are carefully used within the proper intake range, they can be considered a healthy food that contains an abundance of dietary fiber, minerals, and vitamins.

In this study, the number of samples used for monitoring heavy metals and arsenic in seaweed was insufficient to perform an in-depth evaluation of food safety. Therefore, additional studies such as evaluating the risk of inorganic As in seaweeds using internationally authorized methods like BMD and statistical relationships studies between the contents of As species in seaweeds and diverse marine organisms are necessary. These additional approaches will provide useful guidelines for seaweed consumption.