4. MATERIALS AND METHODS
4.2 D ATA COLLECTION
As mentioned in Chapter 4.1, one of the essential phases of risk-benefit assessment is the formulation of the problem, which includes the elaboration of the question of RBA. The elaboration of this question requires the definition of several aspects. Regarding the population of interest, it was stipulated that this would be the adult Portuguese population (over 18 years of age), which covers more than 81% of the population in Portugal (PORDATA, 2021).
In order to assess the risk-benefit of seaweeds as human food, it was considered that the level of aggregation corresponds to a food product (seaweeds). To this assessment, two species were selected for study. The selection of these two species was based on their importance in the market and their current consumption, as well as the availability of studies considering them. Since the most consumed species worldwide are Kappaphycus
Figure 7 – Risk-benefit assessment approach (adapted from Assunção et al. 2019).
alvarezii, Eucheuma denticulatum, Gracilaria verrucosa, Saccharina japonica, Porphyra spp., and Undaria pinnatifida (WBG, 2016), two species were selected from this group. Based on this, studies that specifically evaluated these species and whose samples had been collected in Portugal, were identified:
• Leal et al. (1997), collected the species Enteromorpha spp. and Porphyra spp., on three different beaches in Oporto (Matosinhos, Madalena and Cortegaça) between 1994 and 1995;
• Saraiva (2019), collected for his study the species Undaria pinnatifida, Porphyra linearis, and Porphyra umbilicals, on Buarcos beach, in Figueira da Foz, in 2018;
• Paiva et al. (2014) collected in the island of São Miguel, in the Azores, the species Porphyra sp. in 2007, and Osmundea pinnatifida and Fucus spiralis in 2013.
Therefore, considering the previously defined requirements, the edible seaweeds species selected for the study were Porphyra spp. (Figure 8) and Undaria pinnatifida (Figure 9).
Figura 8 - Undaria pinnatifida (source: Zaixso, H., & Boraso, A. (Eds.), 2014).
Figure 9 - Porphyra tenera (source: FAO, 2021a).
Furthermore, research on these two species revealed interesting characteristics that strengthened their selection for this study. Regarding Porphyra spp., Carmona et al. (2006) alluded to the rapid growth of this seaweed, and emphasised the ability of some species of Porphyra to be good concentrators of nutrients. Concerning Undaria pinnatifida, this is highly adaptable species. In addition to its tolerance to warmer temperatures (Hay & Luckens, 1987), this species is also characterized by its easy development in artificial substrates such as metal, wood, plastic polymers, or paints (Ugarte et al., 2006).
Concerning the construction of scenarios, the first step is to define which reference scenario to establish. For the assessment of the impacts of seaweeds in health and diets, a 4 grams dose (or approximately) is normally considered in several studies (Kolb et al, 2004;
Rosales et al. 2020; Teas et al, 2009; van den Driessche et al, 2019). Therefore, in this study, it was defined that the daily consumption of this dose (4 grams) would correspond to the reference scenario.
In order to compare responses to human needs, two more scenarios were defined. These two scenarios correspond to a lower and a higher value compared to the reference scenario.
Regarding the lowest value (Scenario 1), it was considered the lowest value found in the bibliography for daily consumption, that is, 2 grams (Kolb et al, 2004). Although higher
values were identified in the literature (Teas et al, 2009), the daily consumption of 10 grams was considered for Scenario 2. This choice for the Scenario 2 was made not only because this is a more recurrent value in the literature (Al Amin et al, 2018; Cornish et al, 2015), but also given its closer proximity to the reference scenario (similarly to Reference scenario and Scenario 1). Consequently, the chosen scenarios were:
• Reference scenario: daily consumption of 4 g;
• Scenario 1: daily consumption of 2 g;
• Scenario 2: daily consumption of 10 g.
Once all these aspects were established, it was possible to define the Risk-Benefit question: What will be the impact of the increase in consumption of the seaweeds Undaria sp. and Porphyra sp. in the health of the Portuguese adult population?
After the scenarios and the RBQ have been formulated, the next step is the identification and prioritization of health effects. This step, as mentioned, requires a high degree of literature quality. Therefore, the available data provided by large-scale scientific organizations, such as the World Health Organization and European Food Safety Authority, were used.
In the State of the Art chapter, it was already possible to identify and discuss both adverse and beneficial effects associated with the seaweeds consumption. As previously mentioned, one of the main risks related to the excessive consumption of this food is the high exposure to iodine. According to WHO (2019a), the main effects resulting from repeated overexposure to iodine, through ingestion, are related to the thyroid gland and regulation of thyroid hormone production and secretion. According to the same organization, changes in the thyroid gland may be responsible for various consequences in the endocrine, cardiovascular and pulmonary systems, and many other organs.
It should be noted that seaweeds contain some harmful contaminants to the organism, such as cadmium, inorganic arsenic, lead, and mercury (National Food Institute et al., 2019).
Therefore, some effects resulting from exposure to these contaminants are presented below.
Long-term exposure to Cadmium essentially affects the kidneys, but can also have an impact on the bones (WHO, 2019b). It should also be noted that cadmium is considered a human carcinogen, being classified as Group 1 by the International Agency for Research on Cancer (IARC).
The first consequences of long-term exposure to high levels of Inorganic Arsenic are reflected on the skin, namely through pigmentation and lesions. There are also other effects associated with exposure in these conditions, such as diabetes, conjunctivitis, effects on the renal system, among many others (WHO, 2010a). Like cadmium, inorganic arsenic is also part of IARC Group 1.
According to WHO, Lead is considered a cumulative toxicant, and the health effects caused by exposure to this element can affect several systems, namely the neurological, cardiovascular, renal, and haematological systems (WHO, 2010b).
Finally, mercury is an extremely toxic element for human health, and exposure to this element can trigger consequences for the central and peripheral nervous system. Not only when ingested, but also when inhaled or applied dermally, different mercury compounds can lead to neurological and behavioural disorders, which include symptoms such as tremors, memory loss, and cognitive and motor dysfunction (WHO, 2007)
Regarding the positive effects to seaweeds consumption, these were described in the State of the art chapter. Seaweeds are rich in several nutrients and is often seen as a superfood. This factor can be beneficial as it responds to daily human needs for these same nutrients.
Knowing the main health effects that can result from seaweed consumption, it is also essential to know the nutritional composition of the two particular species under study. In order to understand which components are generally identified in Porphyra sp. and Undaria pinnatifida, food composition tables from several countries were analyzed. Food Composition Tables are composed of data that provide detailed nutritional information on foods, including energy, vitamins, and mineral values (INDDEX, 2021; NIHR, 2021). The need for these tables on a national scale is justified by the fact that the same food/meal may
have different nutrient compositions, something that is due to aspects such as different forms of cultivation, soil, climate, or production practices (Greenfield & Southgate).
According to the same authors, in these tables, data may be presented per 100 g of food or per average serving.
Therefore, the French Food Composition table (ANSES-CIQUAL, 2021a) was used, since it presented not only a more complete identification of the components, but also because its values were closer to those mentioned in two other European tables (Swiss and Dutch). The values referring to the composition of Porphyra sp. and Undaria pinnatifida species from the French Table can be seen in Annex I and Annex II, respectively. It should be noted that there was an attempt to verify the existence of these species/algae in the Portuguese Food Composition Table (PortFIR, 2021), but no results were found.
Knowing the nutritional composition of the two species, it becomes necessary to understand how their components respond to human needs. For this purpose, the dietary reference values (DRV) were consulted. These values propose the amount of a certain nutrient that must be consumed by an individual or population to maintain their health (EFSA, 2019). DRV are fully available online through an EFSA interactive platform called the DRV Finder. Within the Dietary Reference Values, it is possible to identify the adequate intake (AI), the average requirement (AR), population reference intake (PRI), and the tolerable upper intake level (UL), where (EFSA, 2021):
• AI represents the “average nutrient level consumed daily by a typical healthy population that is assumed to be adequate for the population's needs”;
• AR is the “level of a nutrient in the diet that meets the daily needs of half the people in a typical healthy population”;
• PRI is the “intake of a nutrient that is likely to meet the needs of almost all healthy people in a population”;
• UL is the “maximum intake of substances in food, such as nutrients or contaminants, that can be consumed daily over a lifetime without adverse health effects”.
Since the study population is the Portuguese adult population, only the DRVs corresponding to adults were considered (Annex II). Although these DRVs are available for a wide range of nutrients, the study considered only those nutrients that, according to the French Food Composition Table, are present in the two species of algae. After analysing the values of these nutrients, it was possible to verify that some were found in considerable residual amounts (when compared to DRV), and thus were discarded.
Therefore, the following nutrients present in both species were considered:
• Minerals: calcium (Ca), copper (Cu), iron (Fe), iodine (I), magnesium (Mg), phosphorus (P), potassium (K), selenium (Se), sodium (Na), and zinc (Zn);
• Vitamins: B3, B9, C and E;
• Protein;
• Fiber.
Table 3 presents the selected nutrient values for the two species under study, based on the French food composition table. Table 4 shows the dietary reference values for the same nutrients.
It is important to point out that, for certain elements, the DRV can vary not only according to age, but also according to sex, a state of pregnancy or due to the presence of some pathology. Therefore, in this study, for each of these groups, the lowest value was always considered for that nutrient, applying the most conservative approach.
Table 3 - Food composition of Porphyra sp. and Undaria pinnatifida species based on values available in the French Food Composition Table (Source: ANSES-CIQUAL, 2021b;
ANSES-CIQUAL, 2021c). The indicated values correspond to the amount of nutrients present in 100 grams of seaweed.
Table 4 - Dietary reference values of selected nutrients (Source: DRV Finder, 2021).
1 Safe and adequate intake: 2 g/day
2 The UL applies to readily dissociable Mg salts (e.g. chloride, sulphate, aspartate, lactate) and compounds like MgO in food supplements, water or added to foods; does not include Mg naturally present in foods and beverages
Protein (g)
Fiber (g)
Minerals Vitamins
Ca (mg)
Cu (mg)
Fe (mg)
I (μg)
K (mg)
Mg (mg)
Na (mg)
P (mg)
Se (μg)
Zn (mg)
B3 (mg)
B9 (mg)
C (μg)
E (mg) Undaria
pinnatifida 14.1 41.4 1000 0.23 17.2 19100 7140 1110 5170 319 72.5 2.03 6.39 237 28 1.13 Porphyra
sp. 31.5 36.3 318 1.06 37.2 5100 1730 486 1980 518 51.2 4.52 5.82 21.7 57.30 2.87
Protein (g/kg)
Fiber (g)
Minerals Vitamins
Ca (mg)
Cu (mg)
Fe (mg)
I (μg)
K (mg)
Mg (mg)
Na1 (mg)
P (mg)
Se (μg)
Zn (mg)
B3 (mg)
B9 (mg)
C (μg)
E (mg)
AI n.a. 25 n.a. 1.3 n.a. 150 3500 300 n.a. 550 70 n.a. n.a. n.a. n.a. 11
AR 0.66 n.a. 750 n.a. 6 n.a. n.a. n.a. n.a. n.a. n.a. 6.2 1.3 250 80 n.a.
PRI 0.83 n.a. 950 n.a. 6 n.a. n.a. n.a. n.a. n.a. n.a. 7.5 1.6 330 95 n.a.
UL n.a. n.a. 2500 5 n.d. 600 n.d. 2502 n.a. n.d. 300 25 10 n.d. n.d. 300
Once the necessary data to assess the potential benefit resulting from the consumption of seaweeds was assembled, the search for data that could provide answers to the potential risk also caused by this consumption was carried out.
As mentioned, some of the contaminants commonly found in seaweeds and whose high exposure can represent a risk for human health are inorganic arsenic, cadmium, lead, and mercury (National Food Institute et al., 2019). Therefore, literature was resorted to understand which contaminants are frequently found specifically in the species of Porphyra and Undaria pinnatifida. Several studies (Caliceti et al., 2002; Leal et al., 1997; Pérez et al. 2007;
Saraiva, 2019; Smith et al., 2010) report the presence of Lead, Cadmium, Copper, Mercury, and Arsenic in these two species. The values identified in two studies which analyzed samples of Porphyra spp. and Undaria pinnatifida collected in Portugal are shown in Table 5.
Table 5 – Contaminants found in Porphyra and Undaria species, in studies carried out on the Portuguese coast.
Specie Location Pb (µg g-1)
Cd (µg g-1)
Cu (µg g-1)
Hg (µg g-1)
As
(µg g-1) Reference
Porphyra
Matosinhos 2 0.56 9 0.24 -
Leal et al.
(1997)
Madalena 2 0.73 11 0.085 -
Cortegaça 2.8 1.1 7.7 0.075 -
Porphyra linearis
Figueira da
Foz 0.06 1.24 - 0.011 54.87 Saraiva
(2019) Porphyra
umbilicalis
Figueira da
Foz 0.1 0.13 - 0.011 46.69 Saraiva
(2019) Undaria
Pinnatifida
Figueira da
Foz 0.06 0.32 - 0.02 46.79 Saraiva
(2019)
In order to assess the risk associated with these contaminants, there are several types of methodologies recommended by EFSA, which may vary depending on the contaminant under analysis. Therefore, the methodologies used to assess the risk associated with the four elements mentioned are described below.
➢ Lead
To characterize the risk for human health associated with lead, and considering the approach referenced in the “Scientific Opinion on Lead in Food”, developed by EFSA (2010), the Margin of Exposure (MOE) approach was applied. According to EFSA (2021), the margin of exposure is “a tool used in risk assessment to explore safety concerns arising from the presence of a potentially toxic substance in food or animal feed”. The higher the Margin of Exposure value, the lower the associated risk (FAO, 2013b). When considering genotoxic or carcinogenic substances, a MOE of 10000 or higher is of low concern for public health (EFSA, 2015). Concerning non-genotoxic substances, a MOE of 100 or higher generally indicates no concern for public health. It should be noted that, according to EFSA (2010), the International Agency for Research on Cancer (IARC) classified lead compounds as probably carcinogenic to humans (Group 2A). Therefore, this corresponds to the first situation, and it will be considered that a MOE equal or above 10000 will be of low concern for health. In order to assess the effects on the kidneys, the MOE calculation was done using the following equation (FAO, 2013b):
MOE= BMDL10
Estimated Dietary Exposure
,
where BMDL10 stands for Benchmark Dose Lower Confidence Limit and represents the 95% lower confidence limit of the Benchmark Dose (BMD) of 10% extra risk (Sand et al, 2011). The BMD approach can be used for all chemicals in food (EFSA Scientific Committee et al., 2016) and corresponds to the concentration/dose that produces a predetermined change in the response rate of an adverse effect (EPA, 2021).
In turn, the BMDL corresponds to the lower limit of the BMD, being this generally used as a reference point (EFSA Scientific Committee et al., 2016). In the case of Lead, and according to the EFSA report (EFSA, 2010), the BMDL10 is 0.63 µg/kg b.w. per day.
To assess the effects at the cardiovascular level, the calculation of the MOE was made using the previous equation, however considering the BMDL01 instead of the BMDL10. This BMDL01 represents the 95% lower confidence limit of the BMD of 1%
extra risk (EFSA, 2010), and for Lead this takes the value of 1.5 µg/kg b.w. per day (EFSA, 2010).
➢ Inorganic Arsenic
Inorganic Arsenic (iAs) is the best toxicologically characterized form of Total Arsenic, which is also responsible for the associated health effects (National Food Institute et al., 2019), which enhances the need to evaluate this element form in the present work.
In the scientific report of EFSA (2014) about "Dietary exposure to inorganic arsenic in the European population", seaweeds were evaluated as part of the category
“Vegetables and vegetable products”. In this category, only a small portion of the samples contained data concerning the amount of iAs, so when these values were not available, a conversion factor of 70% was applied to total arsenic. However, this conversion factor was not applied to the seaweeds group. For this group, when the seaweed type was specified, a conversion factor of 1% was applied.
In “Scientific Opinion on arsenic in food”, developed by EFSA (EFSA, 2009a), the MOE approach was also used in order to carry out the risk characterization for Inorganic Arsenic. In this case, the BMDL01 was considered for MOE calculation.
Regarding BMDL01 for Inorganic Arsenic, it was established by EFSA that, to perform the risk characterization of this element, a range of values should be used, instead of a single number. The same document defines for this case that the range of values from 0.3 to 8 μg/kg body weight (b.w.) per day for BMDL01 should be considered.
Considering this range of values and dividing them by the Estimated Dietary Exposure of Inorganic Arsenic, it will be possible to calculate the MOE. Again, if this value turns out to be less than 10000, there is a risk to human health associated with consuming the food for the dietary exposure considered in the study. This methodology was also applied to this element in this study.
➢ Cadmium
According to the methodology used in the Scientific Opinion of Cadmium in food (EFSA, 2009b), in order to characterize the risk for human health associated with Cadmium, the Tolerable Weekly Intake (TWI) is used. The TWI corresponds to the maximum intake of a given substance present in food, which can be consumed weekly throughout life, without any risk of adverse health effects (EFSA, 2021 - glossary). EFSA has established a TWI for cadmium of 2.5 μg/kg b.w. per week (EFSA, 2009b).
Therefore, it is considered for this study that if a weekly intake value greater than 2.5 μg/kg b.w. is observed for cadmium, there is a risk of adverse effects on human health.
➢ Mercury
Concerning the risk associated with mercury, the approach is similar to that used for cadmium. EFSA has defined the tolerable weekly intake value of 4 μg /kg b.w. per week for total mercury (EFSA CONTAM Panel, 2012). This means that a weekly consumption higher than this value may represent a risk of adverse effects on human health.
All the calculations were performed using the Microsoft Excel for Microsoft 365 MSO (version 2110 Build 16. 0. 14527. 20270) 64-bit.