The Kelp Laminaria hyperborea as a Bioindicator

During the period of 2015-2017, concentrations of the toxic or essential elements Cd, Hg, Pb, Al, Cr, Fe, Ni, Cu, Zn and As were analysed in the species kelp Laminaria hyperborea in an upwelling and nutrient-rich area at Mausund in Frøya municipality in Trøndelag, Norway. Samples were extracted by HNO3 and analysed using inductively coupled plasma mass spectrometry (ICP-MS). In this study, the mean levels in L. hyperborea were significantly different between all years for the elements Hg, Pb, Cr, Fe, Ni and Zn, and the study indicated a temporal increase in the concentrations of Hg, Pb, Fe and Zn during the three-year period. The study showed high levels of Cd and Hg, according to levels set by the Regulation of fertilisers, etc. of organic origin [1]. According to European feed legislation regarding the maximum permitted levels in feed ingredients and complete feed stuff, there were elevated levels for Cd in 2016, for Hg in 2017 and for As throughout the three-year period. The European food legislation also sets maximum permitted levels for Cd, Pb and Hg in food supplements. The Hg levels in 2017 also exceeded these levels for supplements. The lamina of the species L. hyperborea can be used as a bioindicator for short-term exposure, and the results from this study can be considered to be reference levels.


Introduction
The Government of Norway and The Foundation for Industrial and Technical Research at the Norwegian University of Technology (SINTEF), believe that the seafood industry in Trøndelag in Norway will be six-fold by 2050. SINTEF is one of Europe's largest independent research organisations. The background for this claim is the value creation based on a productive sea in 2050 [2]. Environmental monitoring of seafood will remain important in the coming years with increasing production. Traditionally, the levels of essential or toxic elements in seafood are monitored by taking samples from the meat in fish and crab. A supplement for environmental monitoring of marine resources is to analyse the same elements using samples taken from macroalgae. By analysing toxic or essential elements in macroalgae and comparing the concentrations between years, macroalgae can be used as a bioindicator. A bioindicator is an organism that shows the results of environmental impact. The use of a bioindicator can provide information on a wide range of items, including both organic and inorganic pollutants. Pollutants can be toxic elements, such as trace metals, or essential elements that individuals need, but which give negative consequences if excessive concentrations of them occur. The elements selected for analysis in this study are cadmium (Cd), mercury (Hg), lead (Pb), aluminium (Al), chrome (Cr), iron (Fe), nickel (Ni), copper (Cu), zinc (Zn) and arsenic (As). They are inorganic trace elements, which are either essential or toxic. The selection of these elements was based on elements specified in the Norwegian regulation "Regulation of fertilisers, etc. of organic origin".
The purpose of this regulation (Regulation of fertilisers, etc. of organic origin) is to ensure satisfactory quality of products covered by the regulations, to prevent pollution, health and hygiene disadvantages in the manufacture, storage and use of fertilisers, etc. of organic origin and facilitate the use of these products as a resource. The regulations will also contribute to environmentally management of the soil and to take into account the importance of biodiversity [1]. The elements specified in this regulation are shown in Table  1.
The regulation states maximum limits for the elements Cd, Pb, Hg, Ni, Zn, Cu and Cr. Materials containing these elements are placed within quality classes 0, I, II, and III, where 0 is the best and III is the worst (Table 1).
Maximum permitted levels for the elements As, Cd, Pb and Hg in feed ingredients and complete feedstuff are set by the European feed legislation as follows (for 12% moisture content): As (total) is 40 mg kg -1 in feed materials from seaweed and products thereof, 2

2/4
mg kg -1 in feed and 10 mg kg -1 for fish feed; inorganic As is 2 mg kg -1 in feed ingredients; Cd is 1 mg kg -1 in feed and 1 mg kg -1 in fish feed; Pb is 10 mg kg -1 in feed and 5 mg kg -1 in fish feed; and Hg is 0.2 mg kg -1 in feed and 0.1 mg kg -1 in fish feed. The European food legislation regulates food stuffs and food supplements, and the maximum levels for Cd, Pb and Hg in macroalgae are set at 3.0, 3.0 and 0.1 mg kg -1 (wet weight), respectively [3].
Lately, there has been increasing interest in the cultivation and harvest of wild stocks of macroalgae in Norway. High levels of inorganic arsenic and cadmium have been reported in brown algae, and this content may limit the use of brown algae as food and feed ingredients, but there are no regulations for As in macroalgae used as a food supplement [3]. L. hyperborea is an important species used in the alginate industry [4]. Alginate is mainly used for pharmaceuticals, food and industrial products. Macroalgae grow along the coast of Norway, and the kelp forests include other kelp species, epiphytes and animals at different trophic levels, which use the kelp forest as their nursing and feeding habitat [4]. E.g. sea urchin feeds on L. hyperborea. When edible crab, birds and otter eats sea urchin, toxic elements from L. hyperborea will accumulate in the food chain.
Algae are divided into brown, red and green algae. The macroalgae L. hyperborea is a brown algae that grows below low tide and can grow up to 20 years old. The macroalgae consist of stipe, lamina and hapter (holdfast). The old lamina falls off in winter. Growth occurs in the lower part of the lamina, called the meristem, and the growth zone pushes the lamina upwards. The large algae filter water and nutrients throughout their lamina, stipe and hapter [5]. There are regional differences in the large algal population structure (size and age), which is due to regional differences in the ecological consequences of macroalgae harvesting by trawling. In mid-Norway, there are optimal temperature and light conditions, and L. hyperborea has most rapid stipe growth in this area [4]. When temperature and light are low in the winter season, but nutrient levels are high, L. hyperborea grow. The carbohydrates that are used for growth in winter are built up over the long days in summer [6].
Seaweeds accumulate dissolved trace metals from seawater via several binding sites to metallic elements in the polysaccharides in the cell walls [7]. In Trinidade Island, Brazil, background levels of the elements As, Cd, Cu, Pb, Zn and Hg were measured in brown and red algae, and one finding showed significant differences in Cu, Pb and Zn levels in seaweed from north and south of Trinidade Island, partially because of local hydrodynamics. Higher levels of As and Cd in brown algae, compared to red algae, confirm that brown algae accumulate higher concentrations of toxic trace elements [8]. Scheiner et al. [9] reported concentrations of the elements Al, Ti, Cr, Mn, Fe, Ni, Cu, Zn, As, Rb, Sr, Mo, Ba, Pb and I in four seaweed species (L. digitata, L. hyperborea, S. latissima and A. esculenta) from the Isle of Seil, Scotland. The concentrations were ranged in the order Sr>Fe>As>Al>Zn>Ti. Correlation analysis from L. hyperborea showed a strong correlation between the ash content and As level (r=0.7, p<0.5). There were no significant variations in the metals Na, K, Ca and Mg in L. hyperborea between seasons. Based on their results, concentrations of Al, Cr, Mn, Fe and Ni in L. hyperborea were high in September 2011.
Concentrations of the elements As, Cd, Cr, Pb, Mn, Hg and Se in the large alga Alaria nana, collected in Adak Island, Adak Harbor and Clam Cove in Alaska, showed that the variations (14%-43%) were explained by the total length, part of the plant and location, except for lead. The different parts of the plant that contributed to variation were length for As and Se, location for Hg and part of the plant for As, Cd, Cr and Mn. To use large alga as a bioindicator, these data indicate that selection of the studied part of large alga must be consistent. For long-term exposure for As, Cd, Cr and Mn, it is recommended to use the hapter, and for short-term exposure, the lamina can be used to analyse all metals [10]. Chen et al. [11] recommend continual surveillance of metals in seaweed because of the toxicity of the elements Cd, Hg and Pb. They arranged the elements from red seaweed (Porphyria) and brown seaweed (Laminaria and Undaria) collected in Zhejiang, China, in the following order of concentration: Al>Mn>As>Cu>Cr>Ni>Cd>Se>Pb>Hg. Levels of the elements Cu, Ni, Mn and Cd were higher in red seaweed than in brown seaweed, and there was a moderate positive correlation between Ni and Cr (r=0.59, p<0.05).
In view of the fact that the lamina in L. hyperborea is new every year and reflects the uptake of elements throughout the whole organism, the results from analysis of a selection of segments items, where samples are taken from the meristem, will contribute to environmental monitoring.  L. hyperborea pieces were brought to Mausund Field station prior to sampling from the meristem at lamina. Plant size and age for all species were not determined in this study. In total, 5-8 samples from the meristem, approximately one gram (g) for each sample, was taken from each individual. The samples were immediately frozen in 25 mL polystyrene cups. The samples were analysed using inductively coupled plasma mass spectrometry (ICP-MS) at the NTNU.

Materials and Methods
Nine L. hyperborea individuals, for a total of forty-eight samples, were collected at Mausund during 2012-2015. The concentrations of the ten elements Cd, Hg, Pb, Al, Cr, Fe, Ni, Cu, Zn and As are given in μg/g dry weight.  Statistical tests were performed with IBM SPSS Statistics, Version 25. The set premises in SPSS assumed a normal distribution with skewness -/+3, kurtosis -/+3 and Pearson r -/+1. The data were normally distributed, except for Hg (skewness 3.105). Differences in element concentrations were tested between years for all 10 elements using hypothesis testing performed with independent samples t-tests with p<0.05. Correlation analyses were conducted. To assess the relationship between variables, Pearson's r values were used, and a regression line was used to assess the relationship in a scatter chart. Principal component analysis (PCA) was performed.

Results
L.hyperborea were collected at one location (63°54`38`` N, 8°36`43`` E) in Mausund. The mean concentration, minimum and maximum concentration, median and standard deviation of the analysed elements, Cd, Hg, Pb, Al, Cr, Fe, Ni, Cu, Zn and As during 2012-2015, are presented in Table 2. The concentration in each sample is given in the Supporting Information (SI 1).
The mean concentration of Cd is within quality class I. The other elements are within quality class 0. The maximum concentrations of Cd and Hg are close to quality class II (Table 1).
There were significant differences (p<0.05) ( Table 3)  There were moderate positive correlations between the following elements ( There was a strong positive correlation between Cu and As (r=0.770, p<0.01) (Figure 2).
The PCA showed that Cu and As were clustered, and Fe, Al, Hg and Pb were relatively close together (Figure 3). This clustering may reflect a similar bioaccumulation mechanism in the macroalgal cell wall.

Discussion
Environmental monitoring is regularly conducted to investigate if seafood is exposed to contaminants, including toxic elements, and accumulates these contaminants to levels exceeding regulatory guidelines. Usually, the samples are taken from seafood, e.g., fish fillets     or meat from edible crab. A study from mid-Norway showed elevated levels of Cd in the brown meat from edible crab, (Cancer pagurus), and a temporal increase in the concentrations of the elements Se, Cs, Mn, Zn and As during a four-year period [12]. Macroalgae can be investigated in the same way to monitor the environment. As a bioindicator, macroalgae can be used to compare year-to-year variations but also to confirm the good quality of macroalgae used in pharmaceuticals, food and industrial products. There is concern about toxic elements exceeding regulatory guidelines in seafood and seaweeds, due to potential health risks. Chen et al. [11] suggest that because of the confirmed toxicity of some metals, such as Cd, Pb and Hg, surveillance of metals in seaweeds should be performed continually.
In the present study, the toxic or essential elements Cd, Hg, Pb, Al, Cr, Fe, Ni, Cu, Zn and As were analysed in the species kelp L. hyperborea. Using quality classes for the elements Cd, Pb, Hg, Ni, Zn, Cu and Cr (Table 1)  According to European feed legislation regarding the maximum permitted levels in feed ingredients and complete feed stuffs, there were elevated levels for Cd in 2016, for Hg in 2017 and for As throughout the three-year period.
The levels for Hg in 2017 also exceeded maximum permitted levels for food supplements given by the European food legislation.

Conclusion
Comparing the concentrations of toxic or essential elements in the kelp species L. hyperborea, collected in an upwelling and nutrient-rich area in Mausund, showed that there were between-year (2015-2017) variations in the concentrations of some toxic or essential elements. The mean ranked concentrations in L. hyperborea were significantly different between all years for the elements Hg, Pb, Cr, Fe, Ni and Zn, and there was a temporal increase in the concentrations of Hg, Pb, Fe and Zn during the three-year period. The study showed elevated level for Cd and Hg, according to the 'Regulation of fertilisers, etc. of organic origin'. According to European feed legislation regulating the maximum permitted levels in feed ingredients and complete feed stuff, there were elevated levels for Cd in 2016, Hg in 2017 and for As throughout the three-year period. The European food legislation also states maximum permitted levels for Cd, Pb and Hg in food supplements. The levels of Hg in 2017 exceeded these levels. There was a temporal increase in the concentrations of Hb, Pb, Fe and Zn in 2015-2017, and there were correlations between Cu and As but also Hg, Al, Pb and Fe.
A follow-up study should include samples from the species L. hyperborea at times other than early autumn. Different levels of elements may occur in different periods, especially in the spring when the light is strong and there is great photosynthesis activity. The same analyses should also be conducted in seawater and sediments.
The lamina of the species L. hyperborea can be used as a bioindicator for short-term exposure, and the results from this study can be considered reference levels. Marine biological resources are becoming increasingly important to meet the food demands of the increasing human population, but there have to be revised regulations to determine safe levels of toxic, e.g., As, and essential elements in the environmental monitoring of seafood.