The mean concentrations of arsenic, lead, cadmium, mercury, nickel, and zinc, which were obtained from Mytilus galloprovincialis, were calculated with relative standard deviations. In the study, the comparisons of concentrations of the elements were conducted by considering the legal limits of Türkiye and other countries, which were presented in Table 4.
In the linearity test conducted according to the ICP-MS method, it was determined that As, Pb, Cd, Hg, Ni, and Zn provided acceptable results. Additionally, it was determined that the LOD and LOQ values obtained in this study met the Commission Regulation (EC) No 333/2007, which was demonstrated in Table 2. The recovery analyses provided 92% and 97% values. According to the limit of repeatability (the metrological approach), all the results that were obtained in the study were acceptable. Furthermore, the Horrat values of repeatability and reproductivity in all the concentration levels were below 2, which is in accord with the Commission Regulation No 836/2011. In the study, the DMA80 test was conducted by adopting the US EPA method 7473 (Di Bella et al., 2018). Accordingly, the determination method used for Hg was presented with validation results in Table 2.
In the analyses, the cadmium concentrations in the muscles, gills, and digestive gland of the mussels (M. galloprovincialis) were determined as 0.14 mg kg-1, 0.18 mg kg-1, and 0.67 mg kg-1, respectively, while mercury concentrations were determined as 0.012 mg kg-1, 0.072 mg kg-1, 0.04 mg kg-1, respectively (Table 4, Figure 1). Cadmium is highly toxic to kidney and may accumulate in the human body and lead to kidney dysfunction, skeletal damage and reproductive deficiencies (Bernard 2008). Food is the main source of human intake of cadmium. European (EC 2014) and Serbian legislation (Serbia 2019) set a maximum level of 1 mg/kg for bivalve molluscs. The maximum level of Cd obtained in the present study was 0.67 mg/kg, so below the regulated level. The maximum cadmium level permitted for sea products is 0.1 mgkg-1 according to the Turkish Food Codex (2011). The maximum cadmium level permitted is 0.5 mgkg-1 for FAO (1983), 0.2 mgkg-1 for MAFF (1995) and 0.05 mgkg-1 for EU (2001, 2008). The maximum cadmium level muscle and gills of mussel were found to be lower than legal limits of Turkish standards (2011) and MAFF (1995) but cadmium level in muscle and gills of mussel were found to be higher than legal limits of EU (2001,2008) and Turkish Standards (2011). Cadmium level in mussel’s digestive gland was higher than 0.05 mgkg-1 for EU (2001, 2008), 0.1 mgkg-1 for Turkish standards and 0.2 mgkg-1 MAFF (1995). EC 2006, Cadmium Maximum levels were determined as 0.3 in fish and 0.5 (mg/kg wet weight) in crustaceans. Compared with other studies, Guéguen et al., (2011) found the similar mean level of cadmium (0.15 mg/kg) in mussels from France as in the present investigation. Lehel et al., (2018) determined slightly higher mean values in shellfish marketed in Hungary, up to 0.26 mg/kg. Joksimovic and Stankovic, (2011) reported a higher mean concentration of Cd (0.84 mg/kg) in mussel’s tissue from Montenegro. Franco et al., (2002) found Cd concentrations above the regulated maximum level in Mytilus spp. (3.11 mg/kg d.w.) from the coast of northern Spain. Mercury is considered to be the most toxic heavy metal in the environment. In water, microorganisms convert mercury into methylmercury (MeHg). Consumption of fish and fishery products is the major route of human exposure to methyl mercury (Trasande et al., 2005). Maximum limit for mercury in bivalve molluscs was set at 0.5 mg/kg in European Commission (EC, 2006) and Serbian (Serbia, 2019) legislation. According to these results, the concentrations of mercury in all tissues were below all legal limit values which were 0.5 mgkg-1 for FAO (1983), MAFF (1995), Turkish Standards and 1.0 mgkg-1 for EU (2001). Similarly, EC 2006, the maximum limit of mercury in muscle meat of crabs and crab-like shellfish was determined to be 0.5 mg/kg wet weight.
Compared with other studies, similar results are reported by Joksimovic and Stankovic (2011), where Hg mean concentration in mussels originating from Montenegro was 0.05 mg/kg. Also, Lehel et al., (2018) reported low levels of Hg in shellfish marketed in Hungary. The levels of Hg in mussels from the French and Spanish Mediterranean coast were in the range of 0.02–1.24 and 0.03–2.21 mg/kg d.w., respectively, while in the Adriatic Sea they were 0.02–0.50 mg/kg d.w. (Jovic et al., 2011; Stanković and Jovic, 2012). Lead is a highly toxic and carcinogenic metal. European (EC 2015) and Serbian (Serbia 2019) legislation set a maximum limit of 1.5 mg/kg for Pb, in the meat of bivalve molluscs. In our study, the concentrations of lead in muscles, gills, and digestive gland of mussels (M. galloprovincialis) were determined as 0.24 mg kg-1, 0.25 mg kg-1, 0.79 mg kg-1, respectively (Table 4, Figure 1). Maximum concentration reached 0.79 mg kg-1. Arıcı and Bat (2016) reported similar levels of lead in Mediterranean mussels (Mytilus galloprovincialis) from Turkey, which ranged from 0.05–0.7 mg/kg, as well as Guéguen et al., (2011), who reported 0.49 mg/kg ww for Pb in shellfish of the main marketed species at the bay of the Seine. Lehel et al., (2018) investigated heavy metal concentrations in seafood from a fish market in Hungary and reported a mean value of 0.95 mg/kg in shellfish.
The maximum lead level permitted is 2.0 mgkg-1 for MAFF (1995) and 0.5 mgkg-1 for FAO (1983) and 0.3 mgkg-1 for Turkish standards. According to EC, 2006, lead Maximum levels were determined as 0.3 in fish and 0.5 in crustaceans (mg/kg wet weight). Similarly, Novakov et al. (2021) found the lead content in fresh and frozen mussels to be below the predicted limit with an average of 0.32 mg/kg and 0.21 mg/kg, respectively. They found that the maximum concentration reached 0.74 mg/kg.
The International Agency for Research on Cancer classifies As as a carcinogenic agent for humans in category 1 (Sirot et al., 2009). Most arsenic found in fish and seafood is in the form of organic arsenic, which is less toxic (Francesconi 2010; Julshamn et al., 2012). No maximum level in EU legislation has been established for total arsenic in foods. However, previous Serbian law (Serbia, 2014) set a limit for total arsenic of 2 mg/kg for freshwater and saltwater fish, of 3 mg/kg for products from marine fish and of 12 mg/kg for products of tuna fish. In the analyses, it was determined that the concentrations of arsenic were 1.08 mg kg-1 in muscles, 0.656 mg kg-1 in gills, and 6.87 mg kg-1 in the digestive gland of the mussels (M. galloprovincialis), which were presented in Table 4 and Figure 1. It was determined that the arsenic concentrations in muscle tissues and digestive glands of the mussels in question exceeded the daily limit for acceptable arsenic intake, which was 1.0 mgkg-1. According to the Australia standard and EU (2001), the maximum level permitted for arsenic in fish is 1.0 mgkg-1. Lehel et al. (2018) found a mean total arsenic concentration of 3.01 mg/kg in shellfish. Joksimovic and Stankovic (2011), reported high mean levels of As in mussels from Montenegro (14.7 mg/kg d.w.). The results obtained in the investigation of Stankovic et al. (2011) generally indicated higher values of As in Adriatic mussels, than in mussels originating from the Spanish Mediterranean cost. The high level of As in the mussels of the Adriatic region could be explained by the exhibited high concentrations of As (1–19 mg/kg) found in the sediments of the southern Adriatic (Dolenec et al. 1998). Calculating exposure analysis for As, a PTWI of 15 μg/kg set by JECFA (JECFA-776 1989; JECFA-959 2011) was used. Recently, the WHO has withdrawn this PTWI value and has not established a new one (Lehel et al. 2018; WHO, 2021). In the examination of the heavy metal concentrations in mussel tissues within the framework of the study, it was determined that nickel concentrations were 0.23 mg kg-1 in muscles, 0.81 mg kg-1 in gills, and 2 mg kg-1 in digestive glands of the mussels (M. galloprovincialis), which were demonstrated in Table 4 and Figure 1. However, Turkish standards do not include any information about the acceptable nickel levels in fish samples. In Table 4, EU states the limit to be 1.5 mg kg−1. In our study, it was determined that the nickel levels of the muscle samples were below limits indicated by WHO (2001). However, the nickel levels in the digestive glands exceed all the limits.
In the analyses, it was determined that the zinc concentrations of the mussels were 11.1 mg kg-1 in muscles, 15.337 mg kg-1 in gills, and 42.6 mg kg-1 in digestive glands (M. galloprovincialis). The Turkish Food Codex (Anonymous, 2002) states that the maximum zinc level in sea products is permissible up to 50 mgkg-1 while this limit is 30 mgkg-1 in FAO (1983) and 50 mgkg-1 in MAFF (1995). In our study, it was determined that the nickel levels in the muscle tissues and gills of the mussels were below all the limits. On the other hand, the zinc concentrations in the digestive glands of mussels were determined to be higher than the limits of FAO (1983)’s, which is 30 mgkg-1.
These results prove useful in terms of investigating the chemical quality of certain aquatic products, determining possible risks related to their consumption, and realizing that the accumulation of metals depends on the species, distinct tissues, and aquatic environments. In the current study, significant differences were observed in terms of metal accumulation in various tissues and various species. In the results, it was generally observed that heavy metals existed at low concentrations in the muscle tissues of the species while higher concentrations were observed in gill and digestive gland tissues.
In our study, it was determined that the digestive gland tissues of the mussels were above the limit values for Cd according to the Turkish Standards, MAFF (1995), EU (2001, 2008), and FAO (1983). Also, the digestive gland tissues of the mussels were above the limit values for Pb according to FAO (1983) and Turkish standards. Additionally, the amount of As in the muscle of the mussels was above the limit of the Australia Standard (1998) and the As concentrations in digestive gland tissues of mussels were above the limit of Australia Standard (1998) and Eisler (1994). The Hg concentrations in the muscle, gills, and digestive glands of the mussels were below the limits determined by the Turkish Standards (2011) and FAO (1983). The Zinc levels in the digestive glands of mussels were above the limit value according to FAO (1983). The concentration of the heavy metals observed in various tissues of the mussels in the study could be ranked as muscles < gills < digestive gland.
Table 3. PTWI values of trace elements in the organs of mussels
Element
|
PTWI=Cx0.1 kg per week: as mg/adult
|
|
|
Muscle
|
Gills
|
Digestive gland
|
The reference value for PTWI
|
Pb
|
|
0.024
|
0.025
|
0.079
|
1.75a
|
As
|
|
0.108
|
0.07
|
0.69
|
1.05e
|
Cd
|
|
0.014
|
0.018
|
0.067
|
0.4b
|
Hg
|
|
0.001
|
0.007
|
0.0040
|
0.35a
|
Ni
|
|
0.0233
|
0.08
|
0.199
|
2.45d
|
Zn
|
|
1.11
|
1.53
|
4.26
|
490c
|
aPTWI values retrieved from FAO/WHO (2004), bPTWI values retrieved from FAO/WHO (2010), cPTWI values retrieved from FAO/WHO (2007), dPTWI values retrieved from WHO (1993), ePTWI values retrieved from WHO (1998) for inorganic Arsenic. The results were considered that one person (70 kg) consumed 100 g of mussels in one week (Bilgin and Uluturhan-Suzer, 2017).
The concentration intervals and distributions of the six elements determined by using ICP-MS in mussels were presented in Table 3. The element with the highest mean concentration in the digestive gland of mussels was determined as Zn (42.6 mgkg-1) while the element with the lowest mean concentration in the muscle of mussels was Hg (0.012 mgkg-1). The concentration intervals of the six elements analyzed in mussels were as the following (presented in mgkg-1): Pb (0.24-0.79), As (0.7-6.9), Cd (0.14-0,67), Hg (0,01-0,07), Ni (0.23-2), Zn (11.1-42.6).
The PTWI values were calculated according to an adult individual with 70 kg of weight. According to the results, Pb in muscles and gills of mussels were 1.3% and 1.4% of PTWI, respectively while Pb in the digestive gland were approximately 5% of PTWI. The concentration of As in the muscle of mussels was approximately 10.3% of PTWI while these values were 65.4% and 6.3% of PTWI in the digestive gland and gills of the mussels, respectively. When the concentrations of Cd in mussels were investigated, it was determined that this value was 3.6% of PTWI in muscles, 4.5% of PTWI in gills, and 16.8% of PTWI in the digestive gland. Additionally, the values calculated according to Hg concentration were 0.3% of PTWI in muscles, 2.1% of PTWI in gills, and 1.1% of PTWI in the digestive gland. The values of Ni in mussels were 0.9% of PTWI in muscles, approximately 3.3% of PTWI in gills, and approximately 8.1% of PTWI in the digestive gland. Furthermore, the concentrations of Zn in mussels were 0.2% of PTWI in muscles, 0.8% of PTWI in the digestive gland, and 0.3% of PTWI in gills (Table 3).
Considering these results, it was observed that the highest value belonged to As in the digestive gland of mussels with 65.4% of PTWI. On the other hand, the lowest percentage belonged to Zn with 0.2% of PTWI in muscles and 0.3% of PTWI in gills of the mussels (Table 3).
Table 4. Hg, Cd, As, Pb, Ni and Zn concentrations (mg kg −1) of the M. galloprovincialis obtained from Istanbul, Türkiye, and the comparisons with maximum limits in fisheries in different countries (the results are presented in mean values ± standard deviation on wet weight, n=3)
|
Hg
|
Cd
|
As
|
Pb
|
Ni
|
Zn
|
Muscle
|
0.01±0.001
|
0.14±0.002
|
1.08±0.085
|
0.24±0.0022
|
0.23±0.001
|
11.1±0.856
|
Gills
|
0.07±0.006
|
0.18±0.014
|
0.66±0.002
|
0.25±0.00245
|
0.81±0.0127
|
15.3±0.997
|
Digestive gland
|
0.04±0.001
|
0.67±0.0008
|
6.9±0.02
|
0.79±0.0064
|
2±0.026
|
42.6±1.543
|
Maximum limits of fisheries
|
|
|
FAO(1983)a
|
0.5
|
0.5
|
-
|
0.5
|
-
|
30
|
EC (2006)b
|
0.5
|
0.5
|
0.1-0.3*
|
0.5
|
-
|
-
|
EU (2008)c
|
-
|
0.05-0.1
|
-
|
-
|
-
|
-
|
EC 2015d
|
|
1.5
|
|
|
|
|
Australia Standarde
|
-
|
0.05
|
1.0e, 1.3f
|
-
|
-
|
-
|
MAFF(1995)g
|
0.5
|
0.2
|
-
|
2.0
|
-
|
50
|
Turkish Standardsh
|
0.5
|
0.10
|
-
|
0.30
|
-
|
50
|
a Food and Agriculture Organization of the United Nations, b European Commission (EC) 2006, cEuropean Union (EU) (2008), dEuropean Commission (EC) 2015, eAustralia Standard (Australia New Zealand Food Authority, 1998), f Eisler(1994), gMinistry of Agriculture, Fisheries and Food, 1995; hTurkish Standards (Anonymous, 2008; TFC, 2011).* Maximum limit for foodstuffs such as rice and rice products according to EC 2006.
The results in the current study indicated that As, Pb, Cd, Hg, Ni, and Zn in the gills and digestive gland of M. galloprovincialis exceeded the maximum limits levels (mg kg-1), which were 0.07 for Hg, 0.67 for Cd, 6.87 for As, 0.79 for Pb, 2 for Ni, and 42.6 for Zn. Furthermore, metal concentrations in muscles were below the maximum limits. Therefore, it is suggested that As, Pb, Cd, Hg, Ni, and Zn concentrations in seafood samples (particularly in mussels) should be monitored periodically. In general, if the PTWI is above the maximum limits in food, the exposure to the contaminant element is a potential health concern. Within this framework, the amount of a contaminant that enters the body via food requires a separate evaluation for each kind of food/or food group that is consumed. Finally, the Estimated Daily Intakes of all kinds of food should be included in the evaluations. Considering the totality of diets, mussels contribute variably, but potentially by significant amounts of the toxic elements. In the current study, when the data presented in Table 3 were considered, element concentrations of the samples varied from 0.01 mg kg-1 to 0.07 mg kg-1 Hg, from 0.14 mg kg-1 to 0.67 mg kg-1 Cd, from 0.66 mg kg-1 to 6.9 mg kg-1 As, and from 0.24 mg kg-1 to 0.79 mg kg-1 Pb. When the heavy metal concentrations of mussels (M. galloprovincialis) in the study were analyzed, the concentrations of arsenic in the muscles, gills, and digestive gland of mussels were 1.08 mg kg-1, 0.66 mg kg-1, and 6.9 mg kg-1, respectively. The Turkish Standards do not include any information for the maximum level of arsenic in fisheries (Anonymous, 2002 and 2008). Furthermore, Food Standards Australia New Zealand authority stated that the maximum limit value for arsenic in fisheries consumed by people was 1 mg kg-1 according to age intervals. The US Environmental Protection Agency (USEPA) (1997) stated that Total arsenic concentrations in shellfish tend to be higher than those for finfish. In the data sumarized by Chew ( 1996), average total arsenic concentrations for shellfish ranged from 0.2 to 126 ppm. The highest concentrations were seen in two mollusk samples. However, mollusk values were highly variable among the 20 samples tested (range: 1-126 ppm; Chew, 1996). In the study by Ballin et al. (1994), the total arsenic in 4 species of shellfish ranged from 2.6 to 21 ppm; the highest concentration was found in lobster. The average value from pooled samples of blue mussels ( 40 samples) was 2.6 ppm (Ballin et al., 1994). Lawrence et al. (1986) found the total arsenic in replicate lobster, scallop and shrimp samples to be 5.2 ppm, 0.68 ppm and 20.8 ppm respectively. The concentrations of total arsenic in a samples of lobster purchased commercially in Ottawa was 4.7 ppm and in a shrimp sample was 7.2 ppm. Lopez et al. (1 994) found values of 4.01 ppm, 0.34 ppm and 2.95 ppm for commercially purchased samples of cockles, prawns, and mussels respectively.
When the literature was reviewed, a study conducted in the Bartin and Inebolu Ports, Türkiye, compared M. galloprovincialis samples between the two ports. In this study, the researchers reported that the amounts of As (9.89 mgkg-1, dry weight) and Pb (0.88 mgkg-1, dry weight) from Bartin port were significantly higher than the samples collected from Inebolu port (6.38 and 0.83 mgkg-1, dry weight, respectively). Furthermore, the study reported that the concentrations of Co (0.64 mgkg-1, dry weight), Cu (39.5 mgkg-1, dry weight), and Fe (458.66 mgkg-1, dry weight) were significantly higher than the samples collected from the Bartin port (0.16, 7.05, and 409.84 mgkg-1, dry weight, respectively) (p<0.05). In the comparison of the heavy metal concentrations with the limit values, it was determined that As, Cd, Cu and Zn of M. galloprovincialis in Inebolu port and As, Cd and Zn concentrations in Bartin port exceeded the limit values. This indicated the presence of pollution in terms of heavy metals (Gökkuş & Berber, 2019).
The World Health Organization stated that for all groups of humans, the provisional tolerable weekly intake (PTWI) of Cd and Pb were 5 and 25 µg kg -1 of body weight, respectively. These are equivalent to 350 and 1,750 µg/week, for an adult human weighing 70 kg, respectively (WHO/FAO/IAEA, 1996). The maximum allowance concentrations (MAC) for each food are reported by considering domestic food consumption data. Accordingly, the total amounts of the metal of interest from those food species should not exceed the amounts mentioned above (weekly, 350 µg Cd, 1,75 µg Pb, for an adult human weighing 70 kg). On the other hand, the data for food consumption depends on variables such as income, household consumption, age groups, and socioeconomic factors. Furthermore, it is obvious that diets significantly vary depending on countries.
Furthermore, these results can be related to certain bioaccumulation trends, which are based on different habitats, lifestyles, and diet regimes of crustaceans. Certain studies reported that metal accumulation resulted in capacities that were unique to species for bivalves (Usero et al., 1997; Irato et al., 2003). Additionally, certain researchers reported that these differences were associated with the metabolic rates of bivalves (Najdek & Sapunar, 1987). In the current study, the concentrations of As, Pb, Cd, Ni, and Zn in the digestive gland of the mussels were compared to the concentrations in other tissues. Within this framework, the results pointed out that the toxic materials were accumulated in the digestive glands for storage or detoxification. In terms of bivalves, gut content includes a mixture of biogenic and lithogenic materials. The filter-feeding bivalves uptake metals from specific sources by ingesting suspended particulate matter, which includes planktonic organisms, bacteria, and inorganic particulate matter from the water column. In previous studies, it was reported that the availability of terrigenous inorganic materials, such as Cr, Cd, Al, and Fe, were discovered in foods (Szefer et al. 2004; Giarratano and Amin, 2010). Additionally, variations of metal concentrations based on seasons in bivalves are due to numerous factors such as considerable differences in water temperatures, the stream of particulate metals to coastal waters, food availability due to the transfer of metals from water to filter-feeding organisms, and changes in weight during gonadal development, and release of biomass related to sexual production (Szefer et al., 2004; Giarratano & Amin, 2010).
According to the study conducted by Bilgin and Ulutarhan in Homa Lagoon, M. galloprovincialis had Hg concentrations between 0.095 μg g−1 and 0.154 μg g−1 (dry weight) in soft tissues while having Cd concentrations between 0.22 μg g−1 and 0.51 μg g−1, Pb concentrations between 0.81 μg g−1 and 2.47 μg g−1, and Zn concentrations between 79.8 μg g−1 and 232 μg g−1. The digestive gland of M. galloprovincialis had Hg concentrations between 0.139 μg g−1 and 0.251 μg g−1 (dry weight), Cd concentrations between 0.29 μg g−1 and 0.48 μg g−1, Pb concentrations between 0.99 μg g−1 and 2.77 μg g−1, and Zn concentrations between 61.0 μg g−1 and 147 μg g−1. The maximum Hg concentrations of M. galloprovincialis were respectively discovered in February and May, which are the pre-spawning periods. It was reported that due to the increased feeding and meat weight during gonadal development, higher accumulations of Hg and other chemicals occurred in the pre-spawning periods (Najdek & Sapunar, 1987; Vlahogianni et al., 2007).
In a study by Camilleri et al. (2020), the toxic elements of M. galloprovincialis taken from different regions were investigated. The mean values of heavy metals obtained in this study were below the limits imposed by the European Commission (EC, 2006), which suggested the absence of important risks in the sampling areas. In this study, the values obtained were also significantly lower than the values found by Maanan (2007) in mussel samples obtained from Morocco, which had mean concentrations of Cd, Pb, Hg, Cr, and Mn from 7 to 10 times higher than the values obtained in our study. Additionally, in the current study, the Cd, Pb, Hg, Cr, and Mn values were 3 to 10 times lower than the values reported by the study of Orescanin et al. (2006), which evaluated samples obtained from the Adriatic coasts of Croatia. The highest mercury concentrations were discovered in mussels samples from the Catania coast and these values could be associated with the Etna volcanic activity, which is considered to be one of the major natural sources of mercury release into the environment (Martin et al., 2012). These matters were also confirmed by the highest concentrations of mercury in samples of mussels, which were obtained from the eastern coast of Sicily (Messina, Milazzo, and Syracuse). In the current study, the mean mercury concentrations in all the evaluated tissues of M. galloprovincialis were calculated to be 0.012-0.072 mg kg−1. The tolerable mercury concentration level was determined to be 0.5 mg kg−1 by the Turkish Standards, and FAO (Anonymous, 2008; TFC, 2011; FAO, 1983). In the current study, the concentrations of mercury in the gills of mussels were calculated to be exceeding these legal limits. Mussels and other marine invertebrates are inclined to accumulate pollutants in their tissues (Odẑak et al., 1994; Salvo et al., 2016) while not demonstrating any apparent harmful effects. Probably, this is because these invertebrates respond to the presence of high concentrations of xenobiotic substances. As a result of this study, estimates of both trace metal concentrations in bivalves and international indices (PTWI) indicated that metal accumulation was higher for all metals in the digestive gland than in the gills and muscles. Although the amount of elements in general is below the maximum limits, the amount of consumed mussels should be limited since heavy metals are known to be potentially harmful to human health. It is also necessary to continue monitoring, as it is known that mussels accumulate heavy metals and increase the interest in reducing environmental pollution.