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Selenium and mercury molar ratios in commercial fish from the Baltic Sea: Additional risk assessment criterion for mercury exposure
Food Control 50 (2015) 881e888
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Selenium and mercury molar ratios in commercial fish from the Baltic Sea: Additional risk assessment criterion for mercury exposure Lucyna Polak-Juszczak Department of Food and Environmental Chemistry, National Marine Fisheries Research Institute, ul. Kołłataja 1, Gdynia 81-332, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 August 2014 Received in revised form 14 October 2014 Accepted 26 October 2014 Available online 4 November 2014
Background: Mercury content in fish poses risks to the fish and to those who consume them. The aim of the current study was to verify the protective role of selenium against toxic mercury in Baltic fish. The assessment criteria were the Se:Hg molar ratio, its variability depending on species and tissue, and correlations among Se:Hg ratios, mercury concentration, and specimen length. Assays were performed in muscle and liver tissues of Baltic Sea commercial fish species, i.e., cod, herring, sprat, plaice, and turbot. Results: Mercury concentrations in fish from the Baltic Sea are at low level. The values of the Se:Hg molar ratio were higher than 1 (with the exception of a few cod specimens), with ranges in muscle tissues as follows: cod 0.75e28.2; herring 2.0e50.3; sprat 14.2e56.2; flatfish 1.5e35.1; and in livers as follows: cod 10.9e268.2; herring 10.7e87.1; flatfish 10.2e232. The values of the Se:Hg ratio in muscle and liver tissues were negatively correlated with mercury content and specimen length. Conclusion: Mercury concentrations in commercial fish from the Baltic Sea are low and do not pose risks to consumers. The high values of the Se:Hg molar ratio confirm that mercury levels are safe, and also that selenium might offer protection against methylmercury toxicity, which could increase the safety of fish consumption. The values of the Se:Hg molar ratio could be an additional criterion, in addition to measuring Hg concentrations, for evaluating the risk of mercury exposure in fish from the Baltic Sea. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Fish Risk assessment Molar ratio Se: Hg
1. Introduction Fish are an important source of protein rich in polyunsaturated fatty acids (n-3), vitamins, and minerals that are essential for health and contribute to lower rates of cardiovascular diseases. Fish also contain high levels of essential trace elements, including selenium that, among many functions, plays an antioxidant role and may confer some protection against the most toxic form of mercury, methylmercury (Kaneko & Ralston, 2007; Ralston, 2009; Ralston & Raymond, 2010). This is important since the level of mercury in some fish is high enough that it can have a toxic impact on fish and consumers (Gochfeld, 2003; Hightower & Moore, 2003; Hites et al., 2004). Mercury levels in marine organisms tend to increase with draogo & Amyot, 2013), and this is growth and trophic level (Oue especially true of large fish. This type of species in the Baltic is cod. Mercury occurs in cod muscle tissue at high levels that fluctuate depending on fish length (age). Other fish species (herring, sprat, flatfish) inhabiting the Baltic abundantly also contain high levels of mercury despite the decreasing mercury content trends observed
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in the past few decades (Polak-Juszczak, 2009, 2010, 2013). Mercury toxicity depends on many factors, including its co-occurrence with other metals. One of these is selenium. Mercury binds with selenium into complexes such as selenoprotein [(HgeSe)n]m and the simple, but stable, compound e mercury selenide (HgSe), which both reduce toxicity (Yoneda & Suzuki, 2002). According to Razayi et al. (2012), mercury reacts first with a reduced form of selenite in an equimolar ratio. Species, tissue type, and the chemical forms of selenium and mercury impact the complex detoxification process (Chen, Shih, Chou, & Chou, 2002; Lourdes, Cuvin-Aralar, & Furness, 1991). The protective effect of selenium against methylmercury toxicity has been known for nearly three decades (Choi et al., 2008; Ringdal & Julshamn, 1985). Since then, numerous studies have indicated that selenium supplements counteract the negative effects of methylmercury (Berry & Ralston, 2008; Khan & Wang, 2009; Yang, Chen, Gunn, & Belzile, 2008). These studies also indicated that the acute toxicity of mercury, especially of methymercury, can occur when the Se:Hg molar ratio is less than 1 (Peterson, Ralston, Whanger, Oldfield, & Mosher, 2009; Ralston, 2008, 2009; Ralston & Raymond, 2010; Yang, Chen, & Belzile 2010), which suggests that the molar ratio is the key value (rather than the level of mercury and methylmercury) for risk
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2.2. Analytical methods
Fig. 1. Locations of sampling.
assessment. Selenium not only plays a protective role against toxic mercury, but it is a nutritionally essential trace element that is absolutely required for the activity of 25e35 enzymes with important functions (Afonso et al., 2008; Kaneko & Ralston, 2007). Low levels of selenium are associated with increased coronary heart disease (Seppanen, Soininen, Salonen, Lotjonen, & Laatikainen, 2004), while higher (but not toxic) levels of selenium are associated with lower levels of nonfatal heart attacks (Mozaffarian, 2009). Data on mercury levels in Baltic fish are plentiful, but there are none on selenium levels or especially on the protective role this element plays against toxic mercury. The aim of the current study was to verify the protective role of selenium against toxic mercury in commercial fish species from the Baltic Sea. The evaluation criterion was the Se:Hg molar ratio in muscle and liver tissues. This thesis was verified by determining the dependence of this ratio on mercury concentrations and fish size, and the correlation between selenium and mercury and fish length. 2. Materials and methods 2.1. Study material The study material comprised the fish species that occur most abundantly in the Baltic Sea: cod (Gadus morhua), herring (Clupea harengus), sprat (Sprattus sprattus), and flatfish (including flounder (Platichthys flesus)), plaice (Pleuronectes platessa), and turbot (Scophthalmus maximus) caught in the 2011e2013 period (Fig. 1). In total, 52 cod specimens, 73 herring, 30 sprat, 65 flounder, 51 plaice, and 22 turbot of varying body lengths were collected (Table 1). Samples were taken from the muscles and livers of single specimens.
Mercury content was assayed with the cold vapor atomic absorption method in an AMA 254 mercury analyzer. The analyses were conducted according to the following procedure. Tissue samples of about 100 mg were placed in the combustion chamber of the analyzer where they were dried and then burned at a temperature of 600 C in an oxygen atmosphere. The measurements were conducted as follows programs: fish muscle tissue e drying time 70 s, decomposition time 120 s, waiting time 50 s; fish liver tissue e drying time 100 s, decomposition time 160 s, waiting time 60 s. Each series of analyses was preceded by measurements of mercury in reference materials of a similar matrix with certified mercury content. Analyses of selenium content were conducted on samples that were first mineralized in concentrated nitric acid (65%) and hydrogen peroxide (30%) in a MARS 5 microwave oven. The mineralized solution samples were heated for 3 h to a temperature of 70 C, then 3 ml of solution was collected from each sample and 3 ml of 25% hydrochloric acid was added. Selenium concentration was measured with the atomic absorption method hydride generation technique in an atomic absorption spectrometer coupled with a Fias 200. The reduction solution was 0.2% sodium borohydride. This same procedure was run on reference materials that were assayed prior to each measurement series. 2.3. Quality assurance measures Two replicates per sample were treated and analyzed separately. Each measurement series was preceded by the analysis of standard reference materials: for selenium CRM-422 cod muscle (Commission of the European Communities, Belgium) with a selenium concentration of 1.63 ± 0.07 mg kg1 and SRM 1577b bovine liver (National Institute of Standards and Technology, USA) with a selenium concentration of 0.73 ± 0.06 mg kg1. The mercury reference materials were CRM-422 cod muscle with a mercury concentration of 0.559 ± 0.016 mg kg1 and SRM 1566b oyster tissue (National Institute of Standards and Technology, USA) with a mercury concentration of 0.037 ± 0.001 mg kg1. The results were in good agreement with the certified values. Throughout the validation process, the designated detection limit for mercury was 0.001 mg kg1 and for selenium it was 0.01 mg kg1. 2.4. Statistical analysis Statistical analysis was performed with STATISTICA 8.0 (Stat Soft 2005, version 7). Fish biological parameters and metal concentrations in fish muscle tissues were examined initially for normal
Table 1 Mercury and selenium concentrations in fish species from Baltic Sea. Arithmetic means ± sd, and Spearman correlations coefficient (Rs) of mercury and selenium with length. Species
N
Tissue
Mercury mean ± sd (mg g1 w.w.)
Platichthys flesus
65 58 51 36 22 20 73 72 30 52 52
muscle liver muscle liver muscle liver muscle liver muscle muscle liver
0.070 0.034 0.047 0.034 0.071 0.024 0.043 0.064 0.017 0.052 0.018
Pleuronectes platessa Scophthalmus maximus Clupea harengus Sprattus sprattus Gadus morhua
*p < 0.001; **p < 0.05; NS ¼ Not significant.
± ± ± ± ± ± ± ± ± ± ±
0.01 0.02 0.027 0.021 0.035 0.007 0.031 0.034 0.004 0.088 0.017
Mercury correlation with length Rs*
Selenium mean ± sd (mg g1 w. w.)
0.46 0.41 0.80 0.62 0.58 0.37 0.73 0.62 0.49 0.72 0.67
0.150 0.612 0.133 0.731 0.285 0.874 0.165 0.737 0.174 0.156 0.585
± ± ± ± ± ± ± ± ± ± ±
0.035 0.198 0.048 0.271 0.076 0.265 0.049 0.222 0.036 0.026 0.126
Selenium correlation with length Rs**
Hg and Se correlation Rs**
0.15 (NS) 0.11 (NS) 0.23 0.13 (NS) 0.56 0.34 0.19 0.24 (NS) 0.9 (NS) 0.55 0.7 (NS)
0.19 0.19 0.25 0.39 0.33 0.17(NS) 0.20 0.10 (NS) 0.37 0.24 0.19 (NS)
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distribution and homogeneity of variances used the Shapiro-Wilks test. Almost all of the sample variables were not normally distributed. Then, the non-parametric Spearman's test was performed to determine correlations between selenium and mercury concentrations and fish length, Se:Hg molar ratios. The level of significance was designated at p < 0.05. We calculated Se:Hg molar ratios by dividing the concentration in mg per kg by the molecular weight. For each fish specimen, we divided the selenium concentration (mg kg1) by 78.96 and the mercury concentration (mg kg1) by 200.59, and then we calculated the Se:Hg ratio. The mean Se:Hg molar ratios were calculated from those of each fish specimen. To better describe and integrate Se-specific benefits in relation to potential Hg-exposure risks, the proposed Selenium Health Benefit Value (Se HBV) was calculated as follows:
i h . Se HBV ¼ Se Hg molar ratio x total Se mmol g1 i h . Hg Se molar ratio x total Hg mmol g1
3. Results
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species examined. A correlation between these elements was not noted in most of the livers. 3.2. Differences in Se:Hg molar ratios among fish species Differences in Se:Hg molar ratios among species and between tissues were noted (Table 2). The mean value of this ratio in muscles ranged from 6.7 to 27.1. The highest value was noted in sprat and the lowest in the muscles of cod, herring, and turbot. The values of the Se:Hg molar ratio was greater than 1 in the muscles and livers of all the species examined. The mean values of the of the Se:Hg molar ratio in the livers of the different fish species varied from 42.2 to 117.7, and they were several times higher than the values noted in the muscles. The highest ratios were noted in cod livers followed by those in flatfish, herring, and sprat. The coefficients of the Se:Hg molar ratio in the muscles and livers were strongly, negatively correlated with the mercury concentration, which was indicated by the high value of Spearman's correlation coefficient (Rs; from 0.49 to 0.95) (Fig. 2). This correlation was the strongest in cod in both the muscles and the livers (Rs > 0.9), and it was weaker only in sprat muscles (Rs ¼ 0.49).
3.1. Differences in selenium and mercury concentration among fish species and tissues
3.3. Differences in selenium: mercury molar ratios within fish species
The levels of mercury varied depending on species and tissue type (Table 1). Mercury occurred at statistically significantly higher levels in the muscles than in the livers. The highest mercury contents were noted in cod muscle, especially that of larger specimens (100 and 110 cm), followed by flounder and turbot. Two times less mercury was noted in the muscles of plaice and herring, and four times less in sprat. Mercury concentrations also varied in the livers of the fish examined. The content of this element was the highest in herring livers, while that in the livers of the flatfish and cod was significantly lower. The mercury concentrations in the muscles and livers of the fish examined were positively correlated with specimen length. The mean concentrations of selenium in the muscles of the species examined were at similar levels. Turbot was the exception with its muscles containing more than twice the selenium than did those of the other species. A positive correlation between selenium and specimen length was noted only in turbot and cod muscles, while a weak positive correlation was noted in herring muscles. Selenium concentrations in livers did not differ significantly among species, and they did not change with specimen length. However, positive, but weak, correlations (Rs from 0.19 to 0.37) were noted between selenium and mercury in the muscles of specimens of the
Differences in Se:Hg molar ratio values were noted in the muscles of specimens of the same species, while much greater differences were noted in those in the livers (Table 2). This greatest range of this ratio in muscles was noted in specimens of herring and sprat, while the smallest were noted in cod and flatfish. The Se:Hg molar ratio was less than 1 (0.74) in the muscles of several large cod (exceeding 100 cm). However, this ratio was higher than 1 ranging widely from 1.7 to 268.2 in the livers of all the fish examined. 4. Discussion 4.1. Differences in mercury and selenium levels and Se:Hg molar ratios among species and between tissues The Se:Hg molar ratios differed significantly among the species examined (Table 2). The values depended primarily on mercury concentrations, because selenium occurred at similar levels for the test species (Table 1). Only in turbot was the concentration of selenium significantly higher, which distinguished this species from the other flatfish. Selenium usually varied less because it is an essential trace element that is regulated in body because is toxic at high levels. Concentrations of mercury, however, differed
Table 2 Mercury and selenium concentrations (mmol/g wet weight) and molar ratios Se:Hg in tissues of fish species (arithmetic means ± sd, and range of molar ratio). Species
Length range [cm]
Tissue
Hg m mol g1 w.w. mean ± sd
Platichthys flesus
16e39
Pleuronectes platessa
19e41
Scophthalmus maximus
23e39
Clupea harengus
11e30
Sprattus sprattus
8e15
muscle liver muscle liver muscle liver muscle liver muscle
0.349 0.168 0.236 0.168 0.355 0.121 0.221 0.303 0.085
Gadus morhua
24e110
muscle liver
0.231 ± 0.14 0.091 ± 0.08
± ± ± ± ± ± ± ± ±
0.17 0.10 0.14 0.11 0.17 0.03 0.15 0.18 0.02
Se m mol g1 w.w. mean ± sd
Se:Hg molar ratio mean ± sd
Se:Hg molar ratio range
0.44 2.52 0.61 3.43 0.96 3.36 0.62 3.87 0.46
6.7 ± 3.5 57.3 ± 28.4 9.7 ± 6.9 71.2 ± 40.9 11,1 ± 3.9 101.2 ± 49.5 13.5 ± 9.4 42.2 ± 17.2 27.1 ± 8.2
1.7e17.9 1.7e146.6 1.5e26.4 26.1e222.3 5.3e20.3 36.8e234.1 2.0e50.3 10.7e87.1 14.2e56.2
1.84 ± 0.42 6.56 ± 2.7
12.4 ± 6.5 117.7 ± 57.3
0.74e28.2 10.9e268.2
1.88 7.76 1.68 9.25 3.61 11.07 2.09 10.35 2.20
± ± ± ± ± ± ± ± ±
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Fig. 2. Relationship between Se:Hg molar ratio and mercury concentration in fish from Baltic Sea.
significantly depending on species and tissue. Biological factors that influence mercury levels in fish tissues and include species, feeding habits, fish age (which is often expressed as specimen length). This last parameter is especially important for species among which the adult specimens achieve large sizes, because fish accumulate mercury with age and mainly in the muscles (Bidone, Castilhos, Santos, Souza, & Lacerda, 1997; Burger et al., 2001; Green & Knutzen, 2003; Lange, Royals, & Connor, 1994; Penedo de Pinho et al., 2002; Polak-Juszczak, 2009, 2012). In the current study, this species was cod. The largest specimens of cod (100 and 110 cm) contained mercury at a level of 0.55 mg kg1. The bioaccumulation of mercury in fish occurs through the trophic chain (Downs, Macleod, & Lester, 1998; Polak-Juszczak, 2012; Swanson et al., 2003). The presence of phytoplankton and zooplankton in marine environments affects the transfer of mercury up the food
chain, which is why an important factor impacting the level of this element in fish is feeding habits associated with life strategy. The current study included fish from various levels of the trophic chain that fed on different types of food. Herring (C. harengus) and sprat (S. sprattus) are strict zooplanktivorous pelagic species in the Baltic Sea (Cardinale, Casini, Arrhenius, & Hakansson, 2003; Casini, Cardinale, & Arrhenius, 2004). Flatfish employ a benthic life strategy and feed mostly on molluscs (Macoma balthica) and small crustaceans, but turbot also prey on small fish. Cod have completely different preferences, and their diet comprises small herring, sprat, and crustaceans, mainly Mesidothea entomon. All of these dietary components contain different levels of mercury: small herring and sprat e 0.012e0.020 mg Hg kg1 w.w., Mesidothea entomon e approximately 0.021 mg Hg kg1 w.w., M. balthica e a mean of 0.053 mg Hg kg1w.w. (Polak-Juszczak, 2009, 2012); zooplankton e
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0.006 Hg kg1 w.w. (Nfon et al., 2009), planktone0.002 mg Hg kg1 w.w. (Nfon et al., 2009; Saniewska et al., 2010). Because concentrations of selenium in majority of the species were at similar levels, the molar ratio depended on mercury concentration. The mercury concentrations in the fish examined were inversely proportional to the Se:Hg molar ratio values. Thus, the species with the lowest mercury values, which was sprat, had the highest Se:Hg molar ratio values (Table 2). The Se:Hg molar ratio gradation in the muscles of fish from the Baltic Sea was as follows: sprat > herring > cod > flatfish (turbot > plaice > flounder). Variations in mercury concentrations were largely determinant of this order, and they were inverse to those of the Se:Hg ratios, as follows: sprat < herring < cod < flatfish (plaice < flounder < turbot). Since mercury accumulates primarily in the muscles, the Se:Hg molar ratios in the muscles also reflect the levels of this element in the fish species examined. The Se:Hg molar ratio values in the livers of the fish species examined were in the inverse order to those in the muscles (Table 2). The highest values were noted in the livers of cod, followed by those of flatfish (turbot > plaice > flounder), and them those of herring. The Se:Hg molar ratio values occurred in a wide range from 2 to 268. These high levels resulted from the significantly smaller mercury concentrations and higher selenium concentrations in the livers, which were two to three times higher than in the muscles. No Se:Hg molar ratios lower than 1 were noted in the livers of any of the specimens of the species examined. Mercury concentrations in commercial fish species caught in the Baltic Sea are low. However, high selenium levels result in Se:Hg molar ratios that exceed 1, indicating that they could play a protective role against methylmercury, the toxic form of mercury. Consequently, methylmercury toxicity might be lower. Yang et al. (2008) proposed the involvement of Se in the demethylation of MeHg to form inorganic and less toxic Hg compounds. The Se:Hg ratios close to the unit suggests a higher probability of the activation of the demethylation mechanism in tissues. Information on the precise Se:Hg molar ratio that guarantees protection from methylmercury would provide the consumer with more information when selecting which fish species to purchase. However, it bears posing the questions of to what degree an Se:Hg ratio of more than 1, or even much higher, protects against mercury toxicity, and whether this should be the primary criterion for risk assessment. It is highly unlikely that a single Se:Hg ratio value would be applicable to different species inhabiting different regions. Therefore, studies of mercury toxicity should include selenium levels so as to foster a better understanding of the relationship between these elements. Further research is needed, particularly in isolating and speciation the seleniumemercury complexes, identifying the effects of specimen sizes, and determining the impact of mercury and selenium levels on Se:Hg ratios. 4.2. Selenium: mercury molar ratios within species The results of the studies indicated there were statistically significant differences in the values of the Se:Hg molar ratio among fish species in the Baltic Sea. The Se:Hg molar ratios differed significantly also among specimens of the same species, and their values decreased with fish length (Fig. 3). Mercury mainly impacted this dependence, because, as was mentioned previously, it accumulated with fish age (Green & Knutzen, 2003; Penedo de Pinho et al., 2002; Simonin, Loukmas, Skinner, & Roy, 2008; Storelli, Stuffler, & Marcotrigiano, 2002). Strong positive correlations between mercury concentrations and specimen length were noted in all of the species examined (Table 1). Selenium concentrations only increased with length in turbot and in the muscles of cod (but to a lesser degree than mercury). Fish species with adults of large sizes generally
885
have high levels of mercury, which means that, the value of the Se:Hg molar ratio is low. This was confirmed in the cod examined in the present study (Fig. 3). The Se:Hg molar ratio was lower than 1 (0.74) in large cod specimens (100 and 110 cm), which signals high mercury concentration and risk to consumer health. These fish should be withdrawn from the market by the responsible authorities. It should also be underscored here that such large cod are seldom caught in the Baltic Sea. In the sprat, herring, and flatfish examined, all of which are considered to be small fish (10e30 cm), all specimens had Se:Hg molar ratio values higher than 1 and low level of mercury. The dependence between the Se:Hg molar ratio and mercury concentrations in individual specimens within species was confirmed with the non-parametric Spearman's test presented in Fig. 2. Based on these dependencies, the threshold mercury concentration was determined at which Se:Hg molar ratio was below 1. Ralston and Raymond (2010), who were cited earlier, demonstrated that selenium lowers the toxicity of methylmercury when the Se:Hg molar ratio is higher than 1:1. In our considerations, we adopted an even more rigorous and safer range for the protective effects of selenium, namely a Se:Hg molar ratio exceeding 5:1 (Figs. 2 and 3), the dotted horizontal lines correspond to molar ratios of 5:1On this basis determined the mercury concentration at which molar ratio was above 5. The turbot muscles we examined with a mercury concentration of 0.125 mg kg1 all had Se:Hg molar ratios above the range of 1e5 (Fig. 2). The remaining flatfish flounder and plaice, had ratios above 5, which corresponded to mercury concentrations of up to 0.07 mg kg1. All of the sprat had coefficients above 5. According to this mercury concentrations in herring did not exceed 0.095 mg kg1 while those in cod were substantially higher up to 0.160 mg Hg kg1. The mercury concentration above is within the prescribed EU limit, and is within a range of 14e36 % of it. Moreover, the Se:Hg ratio value of 5 indicates that selenium provides protection against methylmercury toxicity. This does not mean, however, that the selenium in these fish completely eliminated the toxicity of metylmercury, but one can assume that determined the level of mercury had not for health negative results. The Se:Hg molar ratio values in the specimens within the same species were also negatively correlated with length (Fig. 3). Such strong negative correlations occurred within most species, as is evidenced by Spearman's coefficient for muscles (Rs from 0.44 to 0.68) and livers (Rs from 0.40 to 0.90). The preceding correlation was found lacking in both tissue types sampled from turbot. These dependencies were used to determine limits of specimen length at which the molar ratio was above 5 and contents of selenium can play a protective role against the toxicity of methylmercury. The range of the Se:Hg molar ratio was greater than 5 in all lengths of turbot and sprat (Fig. 3). Smaller specimens of the other species had also ratios higher than 5, namely flounder and plaice with lengths to 27 cm, herring measuring up to 26 cm, and cod up to 60 cm. The preceding specimen lengths occur most frequently in commercial catches in the Baltic Sea. The aim of the current study was to determine Se:Hg ratios in fish species from the Baltic Sea, which could be used to assess methylmercury toxicity. However, the differences among the species were too large to permit determining the average Se:Hg ratio value that could be used as a measure of methylmercury toxicity reduction. In summation, the Se:Hg molar ratios in the Baltic fish species from the present study were generally above 1, and in the majority of specimens they exceeded 5. Fish with higher mercury contents had lower molar ratios than fish with low mercury concentrations. As fish size increased, molar ratios decreased and mercury contents increased. Thus, large cod had Se:Hg ratio values of less than 1 while they contained mercury at levels that exceeded present EU limits. Generally, the following can be concluded: Se:Hg molar ratios exceed 1; the levels of mercury in the fish examined are safe and
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Fig. 3. Relationship between Se:Hg molar ratio and length of fish from Baltic Sea.
are below the limits set forth by the EU; the sizes of the fish that predominate in commercial catches are safe with regard to mercury; the possible protective role of selenium against methylmercury toxicity could increase the safety of consuming Baltic Sea fish. The threshold specimen lengths determined above and the threshold concentrations of mercury do not indicate the total reduction of toxic mercury, but rather safe levels. Information regarding the Se:Hg molar ratio at which complete protection from toxic mercury is effected would be very valuable information for risk management. However, the examinations performed did not permit determining the threshold limits. Determining such indicators requires further studies on various fish species, especially of those in which mercury occurs at high levels. 4.3. Risk management, risks to consumers In the European Union, the mercury limit in examined fish is 500 mg kg1. The mean mercury concentration in the muscles of
Baltic fish ranged from 17 mg kg1 in sprat to 70 mg kg1 in the other commercial species, which was from 3.4% to 14% of this limit. However, there were specimens among the species examined, mainly large cod, which contained higher levels of mercury. The mercury content in a few specimens of cod exceeded values of 500 mg kg1. These specimens measured 100 and 110 cm, but this length is rare in commercial catches. However, the mercury content of cod measuring above 60e90 cm in length was more than 150 mg kg1. We would like to reiterate something that was confirmed earlier; namely that cod measuring more than 60 cm do not benefit from the protective role of selenium against mercury. It follows that consuming 100 g of meat from such large specimens (100 cm) delivers 50 mg of mercury, which is 17.9% of the Tolerable Weekly Intake (TWI). The level of TWI is determined by the European Food Safety Authority (EFSA, 2012) and is 280 mg Hg/week for consumers weighing 70 kg. The organic form of mercury, methylmercury is substantially more toxic to humans. In herring and sprat from Baltic Sea
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methylmercury is about 80% of total mercury, in flounder and turbot over 82%, and in cod 86% (results from investigations per ski 2003; Kwasniak formed in our laboratory, Barska and Skrzyn et al., 2012). In a study of fish and seafood from the German market, the average methylmercury proportion of total mercury was 70% (Kubala et al., 2011). The TWI for methylmercury set by the EFSA (2012) (1.3 mg/week per kg body weight) is 91 mg/week for a consumer weighing 70 kg. Assuming that in 100 g of cod contains 43 mg methylmercury, exceeding TWI could occur by consuming 212 g of large cod (100 cm) with a mercury concentration of 500 mg kg1. Thus, the consumption of such large cod poses a threat to consumer health. Many people are aware of the advantages of fish consumption (readily digestible protein, omega-3 fatty acids, minerals), and they consume them frequently in large quantities. For such consumers, it is recommended that they select carefully the species and individual fish destined for consumption. It is always recommended to choose fish with low mercury contents, which, according to simplified consumer advice, means small fish. How our investigations show all fish from Baltic Sea contain safe levels of mercury. However, Se:Hg molar ratios higher than 5 in flatfish up to 27 cm, herring up to 26 cm, cod to 60 cm, and sprat without any limitation can additionally partly reduce the toxicity of methylmercury. Among the Baltic Sea fish examined, values of Se:Hg above 5 were noted in 58% of the flounder, 72% of the flatfish, 83% of the turbot, 91% of the herring, 88% of the cod, and 100% of the sprat (Table 3). This is yet another strong argument to bear in mind when choosing fish for consumption. The results of Hg and Se contents of individual fish from the Baltic Sea were used to calculate the Se HBV (Materials and methods 2.4). The mean of these individual values was used to determine the Se HBV for the studied fish species (Fig. 4). The Se HBV can be useful in differentiating between small and large fish and species that are a rich sources of Se from those that contain excess Hg. All specimens of turbot and sprat, and small specimens of flounder, plaice, and herring (length < 30 cm), had positive Se HBV values ranging from 15 to 55. Positive values of this index were also noted in cod lengths below 60 cm. Large specimens (length > 30 cm) of flounder, plaice, and herring had also positive values of Se HBV, but on a lower level (about 7). However, these values were negative (about e 2.1) in the largest cod exceeding 60 cm in length, which also had Se:Hg ratio values below 1, indicating mercury toxicity. The Se:Hg molar ratio appears to be an additional criterion of risk assessment from mercury exposure in addition to determining mercury levels. However since still, the practical implications of the modification of methylmercury toxicity by selenium are unclear we suggest exercising caution
Table 3 Percent of specimens of each species with molar ratios Se:Hg in range: 0e1; 1e5; >5. Species
Tissue
Se:Hg molar ratios 0e1 (%)
1e5 (%)
>5 (%)
Platichthys flesus
muscle liver muscle liver muscle liver muscle liver muscle muscle liver
e e e e e e e e e 2.6 e
42.0 e 28.4 e 13.3 e 11.0 e e 9.3 e
58.0 100 71.6 100 82.7 100 91.0 100 100 88.0 100
Pleuronectes platessa Scophthalmus maximus Clupea harengus Sprattus sprattus Gadus morhua
887
Fig. 4. Selenium health benefit values Se HBV ¼ [Se/Hg molar ratio total Se (mmol kg1)] [Hg/Se molar ratio total Hg (mmol kg1)] for fish species: turbot for all length of specimens; plaice for length < 30 cm and length > 30 cm, flounder for length < 30 cm and length > 30 cm, herring for length < 27 cm and length > 27 cm, sprat for all length of specimens, cod for length < 60 cm and length > 60 cm.
before Se:Hg ratios become part of risk assessment for mercury toxicity. 5. Conclusions The study results indicate that mercury concentrations in commercial fish from the Baltic Sea are below the current EU limit, and, thus, they do not pose risks to consumers. The low mercury levels were also confirmed by the high Se:Hg molar ratios, which exceeded 1 and were higher than 5 in most of the fish examined. High ratio values were noted primarily in small specimens, and the fish inhabiting the Baltic Sea are primarily small species. From 70 to 90% of the Baltic Sea fish examined had Se:Hg molar ratios that exceeded 5, except for flounder (58%). Only in large cod (greater than 100 cm), which represented 3.8% of the cod examined, was the value of this ratio less than 1. This indicates that the commercial fish in the Baltic Sea have low concentrations of mercury and high Se:Hg values, which guarantee safe fish consumption. The Se:Hg molar ratio could be an additional criterion, along with measuring Hg concentrations, for assessing the risk of mercury exposure from Baltic Sea fish. Considerations of the potential protection of selenium against mercury toxicity might be important when assessing risk to people who consume fish frequently. It will be difficult to communicate this message based on molar ratio values since it requires knowledge of mercury and selenium concentrations in fish of different species and sizes from various regions. Threshold values must be assigned to different human populations. The current study indicates that the range of Se:Hg ratio values within species differ widely, which further complicates assigning threshold values. Therefore, in my opinion, using this ratio at present as the main criterion in mercury toxicity assessment is premature. Acknowledgments The author would like to thank Leszek Barcz and the crew of the r/v Baltica for the collection of fish samples. References Afonso, C., Lourenco, H. M., Pereira, C., Martins, M. F., Carvalho, M. L., Castro, M., et al. (2008). Total and organic mercury, selenium and a-tocopherol in some
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deep-water fish species. Journal of the Science of Food and Agriculture, 88, 2543e2550. Berry, M. J., & Ralston, N. V. C. (2008). Mercury toxicity and the mitigating role of selenium. EcoHealth, 5, 456e459. Bidone, E. D., Castilhos, Z. C., Santos, T. J. S., Souza, T. M. C., & Lacerda, L. D. (1997). Fish contamination and human exposure to mercury in Tartarugalzinho River, Northern Amazon, Brazil: a screening approach. Water Air and Soil Pollution, 97, 9e15. Burger, J., Gaines, K. F., Boring, C. S., Stephens, W. L., Jr., Snodgrass, J., & Gochfeld, M. (2001). Mercury and selenium in fish from the Savannah River: species, trophic level, and location differences. Environmental Research, 87, 108e118. Cardinale, M., Casini, M., Arrhenius, F., & Hakansson, N. (2003). Dial spatial distribution and feeding activity of herring (Clupea harengus) and sprat (Sprattus sprattus) in the Baltic Sea. Aquatic Living Resources, 16, 283e292. Casini, M., Cardinale, M., & Arrhenius, F. (2004). Feeding preferences of herring (Clupea harengus) and sprat (Sprattus sprattus) in the southern Baltic Sea. ICES Journal of Marine Science, 61, 1267e1277. Chen, M. H., Shih, C. C., Chou, C. L., & Chou, L. S. (2002). Mercury organic mercury and selenium in small cetaceans in Taiwanese waters. Marine Pollution Bulletin, 45, 237e245. Choi, A. L., Budtz-Jorgensen, E., Jorgensen, P. J., Steuerwald, U., Debes, F., Weihe, P., et al. (2008). Selenium as a potential protective factor against mercury developmental neurotoxicity. Environmental Research, 107, 45e52. Downs, S. G., Macleod, C. L., & Lester, J. M. (1998). Mercury in precipitation and its relation to bioaccumulation in fish: a literature review. Water Air and Soil Pollution, 108, 149e187. EFSA. (2012). Scientific opinion on the risk for public health related to the presence of mercury and methylmercury in food (CONTAM). EFSA Journal, 10(12), 2985 http://www.efsa.europa.eu/en/efsajournal/doc/2985.pdf Accessed June 2014. Gochfeld, M. (2003). Cases of mercury exposure, bioavailability, and absorption. Ecotoxicology and Environmental Safety, 56, 174e179. Green, N. W., & Knutzen, J. (2003). Organohalogens and metals in marine fish and mussels and some relationships to biological variables at reference localities in Norway. Marine Pollution Bulletin, 46, 362e377. Hightower, J. M., & Moore, D. (2003). Mercury levels in high-end consumers of fish. Environmental Health Perspectives, 111, 604e608. Hites, R. A., Foran, J. A., Carpenter, D. O., Hamilton, M. C., Knuth, B. A., & Schwager, S. J. (2004). Global assessment of organic contaminants in farmed salmon. Science, 303, 226e229. Kaneko, J. J., & Ralston, N. V. C. (2007). Selenium and mercury in pelagic fish in the central north Pacific near Hawaii. Biological Trace Element Research, 119, 242e254. Khan, M. A. K., & Wang, F. (2009). Mercuryeselenium compounds and their toxicological significance toward a molecular understanding of the mercuryeselenium antagonism. Environmental Toxicology and Chemistry, 28, 1567e1577. Kuballa, T., Moellers, M., Schoeberl, K., & Lachenmeier, D. W. (2011). Survey of methylmercury in fish and seafood from the southwestern German market. European Food Research and Technology, 232, 737e742. Lange, T. R., Royals, H. E., & Connor, L. L. (1994). Mercury accumulation in largemouth bass (Micropterus salmoides) in a Florida Lake. Archives Environmental Contamination and Toxicology, 27, 466e471. Lourdes, M. A., Cuvin-Aralar, A., & Furness, R. W. (1991). Mercury and selenium interaction: a review. Ecotoxicology and Environmental Safety, 21, 348e364. Mozaffarian, D. (2009). Fish, mercury, selenium and cardiovascular risk: current evidence and unanswered questions. International Journal of Environmental Research and Public Health, 6, 1894e1916. Nfon, E., Cousins, L. T., J€ aervinen, O., Mukherjee, A. B., Verta, M., & Broman, D. (2009). Trophodynamics of mercury and other trace elements in a pelagic food chain from the Baltic Sea. Science of the Total Environment, 407, 6267e6274.
Penedo de Pinho, A. P., Guimaraes, J. R. D., Marins, A. S., Costa, P. A. S., Olavo, G., & Valentin, J. (2002). Total mercury in muscle tissue of five shark species from Brazilian offshore waters: effects of feeding habit, sex, and length. Environmental Research, 89, 250e258. Peterson, S. A., Ralston, N. V. C., Whanger, P. D., Oldfield, J. E., & Mosher, W. D. (2009). Selenium and mercury interactions with emphasis on fish tissues. Environmental Bioindicator, 4, 318e334. Polak-Juszczak, L. (2009). Temporal trends in the bioaccumulation of trace metals in herring, sprat, and cod from the southern Baltic Sea in the 1994-2003 period. Chemosphere, 76, 1334e1339. Polak-Juszczak, L. (2010). Bioaccumulation and temporal trends of trace elements in flounder from the southern Baltic Sea for the 1996-2003 period. Journal of Toxicology and Environmental Health A, 73(17), 1186e1193. Polak-Juszczak, L. (2012). Bioaccumulation of mercury in the trophic chain of flatfish from the Baltic Sea. Chemosphere, 89, 585e591. Polak-Juszczak, L. (2013). Trace elements in the livers of cod (Gadus morhua L.) from the Baltic Sea: levels and temporal trends. Environmental Monitoring and Assessment, 185(1), 687e694. Quedrago, O., & Amyot, M. (2013). Mercury, arsenic and selenium concentrations in water and fish from Sub-Saharan semi-arid freshwater reservoirs (Burkina Faso). Science of the Total Environment, 444, 243e254. Ralston, N. V. C. (2008). Selenium health benefit values as seafood safety criteria. EcoHealth, 5, 442e455. Ralston, N. V. C. (2009). Introduction to 2nd issue on special topic: selenium and mercury as interactive environmental indicators. Environmental Bioindicator, 4, 286e290. Ralston, N. V. C., & Raymond, L. J. (2010). Dietary selenium's protective effects against methylmercury toxicity. Toxicology, 278, 112e123. Rezayi, M., Esmaeli, A. S., Valinasab, T., & Alavi, J. (2012). Selenium health benefit value (SEHBV) in selected fish from Persian Gulf (Khuzestan). International Journal of Environmental Protection, 2(9), 30e35. Ringdal, O., & Julshamn, K. (1985). Effect of selenite on the uptake of methylmercury in cod (Gadus morhua). Bulletin of Environmental Contamination and Toxicology, 35, 335e344. Saniewska, D., Beldowska, M., Beldowski, J., Saniewski, M., Kwasniak, J., & Falkowska, L. (2010). Distribution of mercury in different environmental compartments in the aquatic ecosystem of the coastal zone of the Southern Baltic Sea. Journal of Environmental Sciences, 22(8), 1144e1150. Seppanen, D., Soininen, P., Salonen, J. T., Lotjonen, S., & Laatikainen, R. (2004). Does mercury promote lipid peroxidation. An in vitro study concerning mercury, copper, and iron in peroxidation of low-density lipoprotein. Biological Trace Element Research, 101, 117e132. Simonin, H. A., Loukmas, J. J., Skinner, L. C., & Roy, K. M. (2008). Lake variability: key factors controlling mercury concentrations in New York state fish. Environmental Pollution, 154, 107e115. Storelli, M. M., Stuffler, R. G., & Marcotrigiano, G. O. (2002). Total and methyl mercury residues in tuna-fish from the Mediterranean Sea. Food Additives and Contaminants, 19, 715e720. Swanson, H. K., Johnston, T. A., Leggett, W. C., Bodaly, R. A., Doucett, R. R., & Cunjak, R. A. (2003). Trophic positions and mercury bioaccumulation in rainbow smelt (Osmerus mordx) and native forage fish in northwestern Ontario Lakes. Ecosystems, 6, 289e299. Yang, D. Y., Chen, Y. W., Gunn, J. M., & Belzile, N. (2008). Selenium and mercury in organisms: interactions and mechanisms. Environmental Research, 16, 71e92. Yang, D. Y., Ye, X., Chen, Y. W., & Belzile, N. (2010). Inverse relationships between selenium and mercury in tissues of young walleye (Stizosedion vitreum) from Canadian boreal lakes. Science of the Total Environment, 408, 1676e1683. Yoneda, S., & Suzuki, K. T. (2002). Detoxification of mercury by selenium by binding of equimolar Hg-Se complex to a specific plasma protein. Pharmacology, 274e280.
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Selenium and mercury molar ratios in commercial fish from the Baltic Sea: Additional risk assessment criterion for mercury exposure Lucyna Polak-Juszczak Department of Food and Environmental Chemistry, National Marine Fisheries Research Institute, ul. Kołłataja 1, Gdynia 81-332, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 August 2014 Received in revised form 14 October 2014 Accepted 26 October 2014 Available online 4 November 2014
Background: Mercury content in fish poses risks to the fish and to those who consume them. The aim of the current study was to verify the protective role of selenium against toxic mercury in Baltic fish. The assessment criteria were the Se:Hg molar ratio, its variability depending on species and tissue, and correlations among Se:Hg ratios, mercury concentration, and specimen length. Assays were performed in muscle and liver tissues of Baltic Sea commercial fish species, i.e., cod, herring, sprat, plaice, and turbot. Results: Mercury concentrations in fish from the Baltic Sea are at low level. The values of the Se:Hg molar ratio were higher than 1 (with the exception of a few cod specimens), with ranges in muscle tissues as follows: cod 0.75e28.2; herring 2.0e50.3; sprat 14.2e56.2; flatfish 1.5e35.1; and in livers as follows: cod 10.9e268.2; herring 10.7e87.1; flatfish 10.2e232. The values of the Se:Hg ratio in muscle and liver tissues were negatively correlated with mercury content and specimen length. Conclusion: Mercury concentrations in commercial fish from the Baltic Sea are low and do not pose risks to consumers. The high values of the Se:Hg molar ratio confirm that mercury levels are safe, and also that selenium might offer protection against methylmercury toxicity, which could increase the safety of fish consumption. The values of the Se:Hg molar ratio could be an additional criterion, in addition to measuring Hg concentrations, for evaluating the risk of mercury exposure in fish from the Baltic Sea. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Fish Risk assessment Molar ratio Se: Hg
1. Introduction Fish are an important source of protein rich in polyunsaturated fatty acids (n-3), vitamins, and minerals that are essential for health and contribute to lower rates of cardiovascular diseases. Fish also contain high levels of essential trace elements, including selenium that, among many functions, plays an antioxidant role and may confer some protection against the most toxic form of mercury, methylmercury (Kaneko & Ralston, 2007; Ralston, 2009; Ralston & Raymond, 2010). This is important since the level of mercury in some fish is high enough that it can have a toxic impact on fish and consumers (Gochfeld, 2003; Hightower & Moore, 2003; Hites et al., 2004). Mercury levels in marine organisms tend to increase with draogo & Amyot, 2013), and this is growth and trophic level (Oue especially true of large fish. This type of species in the Baltic is cod. Mercury occurs in cod muscle tissue at high levels that fluctuate depending on fish length (age). Other fish species (herring, sprat, flatfish) inhabiting the Baltic abundantly also contain high levels of mercury despite the decreasing mercury content trends observed
E-mail address: [email protected]. http://dx.doi.org/10.1016/j.foodcont.2014.10.046 0956-7135/© 2014 Elsevier Ltd. All rights reserved.
in the past few decades (Polak-Juszczak, 2009, 2010, 2013). Mercury toxicity depends on many factors, including its co-occurrence with other metals. One of these is selenium. Mercury binds with selenium into complexes such as selenoprotein [(HgeSe)n]m and the simple, but stable, compound e mercury selenide (HgSe), which both reduce toxicity (Yoneda & Suzuki, 2002). According to Razayi et al. (2012), mercury reacts first with a reduced form of selenite in an equimolar ratio. Species, tissue type, and the chemical forms of selenium and mercury impact the complex detoxification process (Chen, Shih, Chou, & Chou, 2002; Lourdes, Cuvin-Aralar, & Furness, 1991). The protective effect of selenium against methylmercury toxicity has been known for nearly three decades (Choi et al., 2008; Ringdal & Julshamn, 1985). Since then, numerous studies have indicated that selenium supplements counteract the negative effects of methylmercury (Berry & Ralston, 2008; Khan & Wang, 2009; Yang, Chen, Gunn, & Belzile, 2008). These studies also indicated that the acute toxicity of mercury, especially of methymercury, can occur when the Se:Hg molar ratio is less than 1 (Peterson, Ralston, Whanger, Oldfield, & Mosher, 2009; Ralston, 2008, 2009; Ralston & Raymond, 2010; Yang, Chen, & Belzile 2010), which suggests that the molar ratio is the key value (rather than the level of mercury and methylmercury) for risk
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2.2. Analytical methods
Fig. 1. Locations of sampling.
assessment. Selenium not only plays a protective role against toxic mercury, but it is a nutritionally essential trace element that is absolutely required for the activity of 25e35 enzymes with important functions (Afonso et al., 2008; Kaneko & Ralston, 2007). Low levels of selenium are associated with increased coronary heart disease (Seppanen, Soininen, Salonen, Lotjonen, & Laatikainen, 2004), while higher (but not toxic) levels of selenium are associated with lower levels of nonfatal heart attacks (Mozaffarian, 2009). Data on mercury levels in Baltic fish are plentiful, but there are none on selenium levels or especially on the protective role this element plays against toxic mercury. The aim of the current study was to verify the protective role of selenium against toxic mercury in commercial fish species from the Baltic Sea. The evaluation criterion was the Se:Hg molar ratio in muscle and liver tissues. This thesis was verified by determining the dependence of this ratio on mercury concentrations and fish size, and the correlation between selenium and mercury and fish length. 2. Materials and methods 2.1. Study material The study material comprised the fish species that occur most abundantly in the Baltic Sea: cod (Gadus morhua), herring (Clupea harengus), sprat (Sprattus sprattus), and flatfish (including flounder (Platichthys flesus)), plaice (Pleuronectes platessa), and turbot (Scophthalmus maximus) caught in the 2011e2013 period (Fig. 1). In total, 52 cod specimens, 73 herring, 30 sprat, 65 flounder, 51 plaice, and 22 turbot of varying body lengths were collected (Table 1). Samples were taken from the muscles and livers of single specimens.
Mercury content was assayed with the cold vapor atomic absorption method in an AMA 254 mercury analyzer. The analyses were conducted according to the following procedure. Tissue samples of about 100 mg were placed in the combustion chamber of the analyzer where they were dried and then burned at a temperature of 600 C in an oxygen atmosphere. The measurements were conducted as follows programs: fish muscle tissue e drying time 70 s, decomposition time 120 s, waiting time 50 s; fish liver tissue e drying time 100 s, decomposition time 160 s, waiting time 60 s. Each series of analyses was preceded by measurements of mercury in reference materials of a similar matrix with certified mercury content. Analyses of selenium content were conducted on samples that were first mineralized in concentrated nitric acid (65%) and hydrogen peroxide (30%) in a MARS 5 microwave oven. The mineralized solution samples were heated for 3 h to a temperature of 70 C, then 3 ml of solution was collected from each sample and 3 ml of 25% hydrochloric acid was added. Selenium concentration was measured with the atomic absorption method hydride generation technique in an atomic absorption spectrometer coupled with a Fias 200. The reduction solution was 0.2% sodium borohydride. This same procedure was run on reference materials that were assayed prior to each measurement series. 2.3. Quality assurance measures Two replicates per sample were treated and analyzed separately. Each measurement series was preceded by the analysis of standard reference materials: for selenium CRM-422 cod muscle (Commission of the European Communities, Belgium) with a selenium concentration of 1.63 ± 0.07 mg kg1 and SRM 1577b bovine liver (National Institute of Standards and Technology, USA) with a selenium concentration of 0.73 ± 0.06 mg kg1. The mercury reference materials were CRM-422 cod muscle with a mercury concentration of 0.559 ± 0.016 mg kg1 and SRM 1566b oyster tissue (National Institute of Standards and Technology, USA) with a mercury concentration of 0.037 ± 0.001 mg kg1. The results were in good agreement with the certified values. Throughout the validation process, the designated detection limit for mercury was 0.001 mg kg1 and for selenium it was 0.01 mg kg1. 2.4. Statistical analysis Statistical analysis was performed with STATISTICA 8.0 (Stat Soft 2005, version 7). Fish biological parameters and metal concentrations in fish muscle tissues were examined initially for normal
Table 1 Mercury and selenium concentrations in fish species from Baltic Sea. Arithmetic means ± sd, and Spearman correlations coefficient (Rs) of mercury and selenium with length. Species
N
Tissue
Mercury mean ± sd (mg g1 w.w.)
Platichthys flesus
65 58 51 36 22 20 73 72 30 52 52
muscle liver muscle liver muscle liver muscle liver muscle muscle liver
0.070 0.034 0.047 0.034 0.071 0.024 0.043 0.064 0.017 0.052 0.018
Pleuronectes platessa Scophthalmus maximus Clupea harengus Sprattus sprattus Gadus morhua
*p < 0.001; **p < 0.05; NS ¼ Not significant.
± ± ± ± ± ± ± ± ± ± ±
0.01 0.02 0.027 0.021 0.035 0.007 0.031 0.034 0.004 0.088 0.017
Mercury correlation with length Rs*
Selenium mean ± sd (mg g1 w. w.)
0.46 0.41 0.80 0.62 0.58 0.37 0.73 0.62 0.49 0.72 0.67
0.150 0.612 0.133 0.731 0.285 0.874 0.165 0.737 0.174 0.156 0.585
± ± ± ± ± ± ± ± ± ± ±
0.035 0.198 0.048 0.271 0.076 0.265 0.049 0.222 0.036 0.026 0.126
Selenium correlation with length Rs**
Hg and Se correlation Rs**
0.15 (NS) 0.11 (NS) 0.23 0.13 (NS) 0.56 0.34 0.19 0.24 (NS) 0.9 (NS) 0.55 0.7 (NS)
0.19 0.19 0.25 0.39 0.33 0.17(NS) 0.20 0.10 (NS) 0.37 0.24 0.19 (NS)
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distribution and homogeneity of variances used the Shapiro-Wilks test. Almost all of the sample variables were not normally distributed. Then, the non-parametric Spearman's test was performed to determine correlations between selenium and mercury concentrations and fish length, Se:Hg molar ratios. The level of significance was designated at p < 0.05. We calculated Se:Hg molar ratios by dividing the concentration in mg per kg by the molecular weight. For each fish specimen, we divided the selenium concentration (mg kg1) by 78.96 and the mercury concentration (mg kg1) by 200.59, and then we calculated the Se:Hg ratio. The mean Se:Hg molar ratios were calculated from those of each fish specimen. To better describe and integrate Se-specific benefits in relation to potential Hg-exposure risks, the proposed Selenium Health Benefit Value (Se HBV) was calculated as follows:
i h . Se HBV ¼ Se Hg molar ratio x total Se mmol g1 i h . Hg Se molar ratio x total Hg mmol g1
3. Results
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species examined. A correlation between these elements was not noted in most of the livers. 3.2. Differences in Se:Hg molar ratios among fish species Differences in Se:Hg molar ratios among species and between tissues were noted (Table 2). The mean value of this ratio in muscles ranged from 6.7 to 27.1. The highest value was noted in sprat and the lowest in the muscles of cod, herring, and turbot. The values of the Se:Hg molar ratio was greater than 1 in the muscles and livers of all the species examined. The mean values of the of the Se:Hg molar ratio in the livers of the different fish species varied from 42.2 to 117.7, and they were several times higher than the values noted in the muscles. The highest ratios were noted in cod livers followed by those in flatfish, herring, and sprat. The coefficients of the Se:Hg molar ratio in the muscles and livers were strongly, negatively correlated with the mercury concentration, which was indicated by the high value of Spearman's correlation coefficient (Rs; from 0.49 to 0.95) (Fig. 2). This correlation was the strongest in cod in both the muscles and the livers (Rs > 0.9), and it was weaker only in sprat muscles (Rs ¼ 0.49).
3.1. Differences in selenium and mercury concentration among fish species and tissues
3.3. Differences in selenium: mercury molar ratios within fish species
The levels of mercury varied depending on species and tissue type (Table 1). Mercury occurred at statistically significantly higher levels in the muscles than in the livers. The highest mercury contents were noted in cod muscle, especially that of larger specimens (100 and 110 cm), followed by flounder and turbot. Two times less mercury was noted in the muscles of plaice and herring, and four times less in sprat. Mercury concentrations also varied in the livers of the fish examined. The content of this element was the highest in herring livers, while that in the livers of the flatfish and cod was significantly lower. The mercury concentrations in the muscles and livers of the fish examined were positively correlated with specimen length. The mean concentrations of selenium in the muscles of the species examined were at similar levels. Turbot was the exception with its muscles containing more than twice the selenium than did those of the other species. A positive correlation between selenium and specimen length was noted only in turbot and cod muscles, while a weak positive correlation was noted in herring muscles. Selenium concentrations in livers did not differ significantly among species, and they did not change with specimen length. However, positive, but weak, correlations (Rs from 0.19 to 0.37) were noted between selenium and mercury in the muscles of specimens of the
Differences in Se:Hg molar ratio values were noted in the muscles of specimens of the same species, while much greater differences were noted in those in the livers (Table 2). This greatest range of this ratio in muscles was noted in specimens of herring and sprat, while the smallest were noted in cod and flatfish. The Se:Hg molar ratio was less than 1 (0.74) in the muscles of several large cod (exceeding 100 cm). However, this ratio was higher than 1 ranging widely from 1.7 to 268.2 in the livers of all the fish examined. 4. Discussion 4.1. Differences in mercury and selenium levels and Se:Hg molar ratios among species and between tissues The Se:Hg molar ratios differed significantly among the species examined (Table 2). The values depended primarily on mercury concentrations, because selenium occurred at similar levels for the test species (Table 1). Only in turbot was the concentration of selenium significantly higher, which distinguished this species from the other flatfish. Selenium usually varied less because it is an essential trace element that is regulated in body because is toxic at high levels. Concentrations of mercury, however, differed
Table 2 Mercury and selenium concentrations (mmol/g wet weight) and molar ratios Se:Hg in tissues of fish species (arithmetic means ± sd, and range of molar ratio). Species
Length range [cm]
Tissue
Hg m mol g1 w.w. mean ± sd
Platichthys flesus
16e39
Pleuronectes platessa
19e41
Scophthalmus maximus
23e39
Clupea harengus
11e30
Sprattus sprattus
8e15
muscle liver muscle liver muscle liver muscle liver muscle
0.349 0.168 0.236 0.168 0.355 0.121 0.221 0.303 0.085
Gadus morhua
24e110
muscle liver
0.231 ± 0.14 0.091 ± 0.08
± ± ± ± ± ± ± ± ±
0.17 0.10 0.14 0.11 0.17 0.03 0.15 0.18 0.02
Se m mol g1 w.w. mean ± sd
Se:Hg molar ratio mean ± sd
Se:Hg molar ratio range
0.44 2.52 0.61 3.43 0.96 3.36 0.62 3.87 0.46
6.7 ± 3.5 57.3 ± 28.4 9.7 ± 6.9 71.2 ± 40.9 11,1 ± 3.9 101.2 ± 49.5 13.5 ± 9.4 42.2 ± 17.2 27.1 ± 8.2
1.7e17.9 1.7e146.6 1.5e26.4 26.1e222.3 5.3e20.3 36.8e234.1 2.0e50.3 10.7e87.1 14.2e56.2
1.84 ± 0.42 6.56 ± 2.7
12.4 ± 6.5 117.7 ± 57.3
0.74e28.2 10.9e268.2
1.88 7.76 1.68 9.25 3.61 11.07 2.09 10.35 2.20
± ± ± ± ± ± ± ± ±
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Fig. 2. Relationship between Se:Hg molar ratio and mercury concentration in fish from Baltic Sea.
significantly depending on species and tissue. Biological factors that influence mercury levels in fish tissues and include species, feeding habits, fish age (which is often expressed as specimen length). This last parameter is especially important for species among which the adult specimens achieve large sizes, because fish accumulate mercury with age and mainly in the muscles (Bidone, Castilhos, Santos, Souza, & Lacerda, 1997; Burger et al., 2001; Green & Knutzen, 2003; Lange, Royals, & Connor, 1994; Penedo de Pinho et al., 2002; Polak-Juszczak, 2009, 2012). In the current study, this species was cod. The largest specimens of cod (100 and 110 cm) contained mercury at a level of 0.55 mg kg1. The bioaccumulation of mercury in fish occurs through the trophic chain (Downs, Macleod, & Lester, 1998; Polak-Juszczak, 2012; Swanson et al., 2003). The presence of phytoplankton and zooplankton in marine environments affects the transfer of mercury up the food
chain, which is why an important factor impacting the level of this element in fish is feeding habits associated with life strategy. The current study included fish from various levels of the trophic chain that fed on different types of food. Herring (C. harengus) and sprat (S. sprattus) are strict zooplanktivorous pelagic species in the Baltic Sea (Cardinale, Casini, Arrhenius, & Hakansson, 2003; Casini, Cardinale, & Arrhenius, 2004). Flatfish employ a benthic life strategy and feed mostly on molluscs (Macoma balthica) and small crustaceans, but turbot also prey on small fish. Cod have completely different preferences, and their diet comprises small herring, sprat, and crustaceans, mainly Mesidothea entomon. All of these dietary components contain different levels of mercury: small herring and sprat e 0.012e0.020 mg Hg kg1 w.w., Mesidothea entomon e approximately 0.021 mg Hg kg1 w.w., M. balthica e a mean of 0.053 mg Hg kg1w.w. (Polak-Juszczak, 2009, 2012); zooplankton e
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0.006 Hg kg1 w.w. (Nfon et al., 2009), planktone0.002 mg Hg kg1 w.w. (Nfon et al., 2009; Saniewska et al., 2010). Because concentrations of selenium in majority of the species were at similar levels, the molar ratio depended on mercury concentration. The mercury concentrations in the fish examined were inversely proportional to the Se:Hg molar ratio values. Thus, the species with the lowest mercury values, which was sprat, had the highest Se:Hg molar ratio values (Table 2). The Se:Hg molar ratio gradation in the muscles of fish from the Baltic Sea was as follows: sprat > herring > cod > flatfish (turbot > plaice > flounder). Variations in mercury concentrations were largely determinant of this order, and they were inverse to those of the Se:Hg ratios, as follows: sprat < herring < cod < flatfish (plaice < flounder < turbot). Since mercury accumulates primarily in the muscles, the Se:Hg molar ratios in the muscles also reflect the levels of this element in the fish species examined. The Se:Hg molar ratio values in the livers of the fish species examined were in the inverse order to those in the muscles (Table 2). The highest values were noted in the livers of cod, followed by those of flatfish (turbot > plaice > flounder), and them those of herring. The Se:Hg molar ratio values occurred in a wide range from 2 to 268. These high levels resulted from the significantly smaller mercury concentrations and higher selenium concentrations in the livers, which were two to three times higher than in the muscles. No Se:Hg molar ratios lower than 1 were noted in the livers of any of the specimens of the species examined. Mercury concentrations in commercial fish species caught in the Baltic Sea are low. However, high selenium levels result in Se:Hg molar ratios that exceed 1, indicating that they could play a protective role against methylmercury, the toxic form of mercury. Consequently, methylmercury toxicity might be lower. Yang et al. (2008) proposed the involvement of Se in the demethylation of MeHg to form inorganic and less toxic Hg compounds. The Se:Hg ratios close to the unit suggests a higher probability of the activation of the demethylation mechanism in tissues. Information on the precise Se:Hg molar ratio that guarantees protection from methylmercury would provide the consumer with more information when selecting which fish species to purchase. However, it bears posing the questions of to what degree an Se:Hg ratio of more than 1, or even much higher, protects against mercury toxicity, and whether this should be the primary criterion for risk assessment. It is highly unlikely that a single Se:Hg ratio value would be applicable to different species inhabiting different regions. Therefore, studies of mercury toxicity should include selenium levels so as to foster a better understanding of the relationship between these elements. Further research is needed, particularly in isolating and speciation the seleniumemercury complexes, identifying the effects of specimen sizes, and determining the impact of mercury and selenium levels on Se:Hg ratios. 4.2. Selenium: mercury molar ratios within species The results of the studies indicated there were statistically significant differences in the values of the Se:Hg molar ratio among fish species in the Baltic Sea. The Se:Hg molar ratios differed significantly also among specimens of the same species, and their values decreased with fish length (Fig. 3). Mercury mainly impacted this dependence, because, as was mentioned previously, it accumulated with fish age (Green & Knutzen, 2003; Penedo de Pinho et al., 2002; Simonin, Loukmas, Skinner, & Roy, 2008; Storelli, Stuffler, & Marcotrigiano, 2002). Strong positive correlations between mercury concentrations and specimen length were noted in all of the species examined (Table 1). Selenium concentrations only increased with length in turbot and in the muscles of cod (but to a lesser degree than mercury). Fish species with adults of large sizes generally
885
have high levels of mercury, which means that, the value of the Se:Hg molar ratio is low. This was confirmed in the cod examined in the present study (Fig. 3). The Se:Hg molar ratio was lower than 1 (0.74) in large cod specimens (100 and 110 cm), which signals high mercury concentration and risk to consumer health. These fish should be withdrawn from the market by the responsible authorities. It should also be underscored here that such large cod are seldom caught in the Baltic Sea. In the sprat, herring, and flatfish examined, all of which are considered to be small fish (10e30 cm), all specimens had Se:Hg molar ratio values higher than 1 and low level of mercury. The dependence between the Se:Hg molar ratio and mercury concentrations in individual specimens within species was confirmed with the non-parametric Spearman's test presented in Fig. 2. Based on these dependencies, the threshold mercury concentration was determined at which Se:Hg molar ratio was below 1. Ralston and Raymond (2010), who were cited earlier, demonstrated that selenium lowers the toxicity of methylmercury when the Se:Hg molar ratio is higher than 1:1. In our considerations, we adopted an even more rigorous and safer range for the protective effects of selenium, namely a Se:Hg molar ratio exceeding 5:1 (Figs. 2 and 3), the dotted horizontal lines correspond to molar ratios of 5:1On this basis determined the mercury concentration at which molar ratio was above 5. The turbot muscles we examined with a mercury concentration of 0.125 mg kg1 all had Se:Hg molar ratios above the range of 1e5 (Fig. 2). The remaining flatfish flounder and plaice, had ratios above 5, which corresponded to mercury concentrations of up to 0.07 mg kg1. All of the sprat had coefficients above 5. According to this mercury concentrations in herring did not exceed 0.095 mg kg1 while those in cod were substantially higher up to 0.160 mg Hg kg1. The mercury concentration above is within the prescribed EU limit, and is within a range of 14e36 % of it. Moreover, the Se:Hg ratio value of 5 indicates that selenium provides protection against methylmercury toxicity. This does not mean, however, that the selenium in these fish completely eliminated the toxicity of metylmercury, but one can assume that determined the level of mercury had not for health negative results. The Se:Hg molar ratio values in the specimens within the same species were also negatively correlated with length (Fig. 3). Such strong negative correlations occurred within most species, as is evidenced by Spearman's coefficient for muscles (Rs from 0.44 to 0.68) and livers (Rs from 0.40 to 0.90). The preceding correlation was found lacking in both tissue types sampled from turbot. These dependencies were used to determine limits of specimen length at which the molar ratio was above 5 and contents of selenium can play a protective role against the toxicity of methylmercury. The range of the Se:Hg molar ratio was greater than 5 in all lengths of turbot and sprat (Fig. 3). Smaller specimens of the other species had also ratios higher than 5, namely flounder and plaice with lengths to 27 cm, herring measuring up to 26 cm, and cod up to 60 cm. The preceding specimen lengths occur most frequently in commercial catches in the Baltic Sea. The aim of the current study was to determine Se:Hg ratios in fish species from the Baltic Sea, which could be used to assess methylmercury toxicity. However, the differences among the species were too large to permit determining the average Se:Hg ratio value that could be used as a measure of methylmercury toxicity reduction. In summation, the Se:Hg molar ratios in the Baltic fish species from the present study were generally above 1, and in the majority of specimens they exceeded 5. Fish with higher mercury contents had lower molar ratios than fish with low mercury concentrations. As fish size increased, molar ratios decreased and mercury contents increased. Thus, large cod had Se:Hg ratio values of less than 1 while they contained mercury at levels that exceeded present EU limits. Generally, the following can be concluded: Se:Hg molar ratios exceed 1; the levels of mercury in the fish examined are safe and
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Fig. 3. Relationship between Se:Hg molar ratio and length of fish from Baltic Sea.
are below the limits set forth by the EU; the sizes of the fish that predominate in commercial catches are safe with regard to mercury; the possible protective role of selenium against methylmercury toxicity could increase the safety of consuming Baltic Sea fish. The threshold specimen lengths determined above and the threshold concentrations of mercury do not indicate the total reduction of toxic mercury, but rather safe levels. Information regarding the Se:Hg molar ratio at which complete protection from toxic mercury is effected would be very valuable information for risk management. However, the examinations performed did not permit determining the threshold limits. Determining such indicators requires further studies on various fish species, especially of those in which mercury occurs at high levels. 4.3. Risk management, risks to consumers In the European Union, the mercury limit in examined fish is 500 mg kg1. The mean mercury concentration in the muscles of
Baltic fish ranged from 17 mg kg1 in sprat to 70 mg kg1 in the other commercial species, which was from 3.4% to 14% of this limit. However, there were specimens among the species examined, mainly large cod, which contained higher levels of mercury. The mercury content in a few specimens of cod exceeded values of 500 mg kg1. These specimens measured 100 and 110 cm, but this length is rare in commercial catches. However, the mercury content of cod measuring above 60e90 cm in length was more than 150 mg kg1. We would like to reiterate something that was confirmed earlier; namely that cod measuring more than 60 cm do not benefit from the protective role of selenium against mercury. It follows that consuming 100 g of meat from such large specimens (100 cm) delivers 50 mg of mercury, which is 17.9% of the Tolerable Weekly Intake (TWI). The level of TWI is determined by the European Food Safety Authority (EFSA, 2012) and is 280 mg Hg/week for consumers weighing 70 kg. The organic form of mercury, methylmercury is substantially more toxic to humans. In herring and sprat from Baltic Sea
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methylmercury is about 80% of total mercury, in flounder and turbot over 82%, and in cod 86% (results from investigations per ski 2003; Kwasniak formed in our laboratory, Barska and Skrzyn et al., 2012). In a study of fish and seafood from the German market, the average methylmercury proportion of total mercury was 70% (Kubala et al., 2011). The TWI for methylmercury set by the EFSA (2012) (1.3 mg/week per kg body weight) is 91 mg/week for a consumer weighing 70 kg. Assuming that in 100 g of cod contains 43 mg methylmercury, exceeding TWI could occur by consuming 212 g of large cod (100 cm) with a mercury concentration of 500 mg kg1. Thus, the consumption of such large cod poses a threat to consumer health. Many people are aware of the advantages of fish consumption (readily digestible protein, omega-3 fatty acids, minerals), and they consume them frequently in large quantities. For such consumers, it is recommended that they select carefully the species and individual fish destined for consumption. It is always recommended to choose fish with low mercury contents, which, according to simplified consumer advice, means small fish. How our investigations show all fish from Baltic Sea contain safe levels of mercury. However, Se:Hg molar ratios higher than 5 in flatfish up to 27 cm, herring up to 26 cm, cod to 60 cm, and sprat without any limitation can additionally partly reduce the toxicity of methylmercury. Among the Baltic Sea fish examined, values of Se:Hg above 5 were noted in 58% of the flounder, 72% of the flatfish, 83% of the turbot, 91% of the herring, 88% of the cod, and 100% of the sprat (Table 3). This is yet another strong argument to bear in mind when choosing fish for consumption. The results of Hg and Se contents of individual fish from the Baltic Sea were used to calculate the Se HBV (Materials and methods 2.4). The mean of these individual values was used to determine the Se HBV for the studied fish species (Fig. 4). The Se HBV can be useful in differentiating between small and large fish and species that are a rich sources of Se from those that contain excess Hg. All specimens of turbot and sprat, and small specimens of flounder, plaice, and herring (length < 30 cm), had positive Se HBV values ranging from 15 to 55. Positive values of this index were also noted in cod lengths below 60 cm. Large specimens (length > 30 cm) of flounder, plaice, and herring had also positive values of Se HBV, but on a lower level (about 7). However, these values were negative (about e 2.1) in the largest cod exceeding 60 cm in length, which also had Se:Hg ratio values below 1, indicating mercury toxicity. The Se:Hg molar ratio appears to be an additional criterion of risk assessment from mercury exposure in addition to determining mercury levels. However since still, the practical implications of the modification of methylmercury toxicity by selenium are unclear we suggest exercising caution
Table 3 Percent of specimens of each species with molar ratios Se:Hg in range: 0e1; 1e5; >5. Species
Tissue
Se:Hg molar ratios 0e1 (%)
1e5 (%)
>5 (%)
Platichthys flesus
muscle liver muscle liver muscle liver muscle liver muscle muscle liver
e e e e e e e e e 2.6 e
42.0 e 28.4 e 13.3 e 11.0 e e 9.3 e
58.0 100 71.6 100 82.7 100 91.0 100 100 88.0 100
Pleuronectes platessa Scophthalmus maximus Clupea harengus Sprattus sprattus Gadus morhua
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Fig. 4. Selenium health benefit values Se HBV ¼ [Se/Hg molar ratio total Se (mmol kg1)] [Hg/Se molar ratio total Hg (mmol kg1)] for fish species: turbot for all length of specimens; plaice for length < 30 cm and length > 30 cm, flounder for length < 30 cm and length > 30 cm, herring for length < 27 cm and length > 27 cm, sprat for all length of specimens, cod for length < 60 cm and length > 60 cm.
before Se:Hg ratios become part of risk assessment for mercury toxicity. 5. Conclusions The study results indicate that mercury concentrations in commercial fish from the Baltic Sea are below the current EU limit, and, thus, they do not pose risks to consumers. The low mercury levels were also confirmed by the high Se:Hg molar ratios, which exceeded 1 and were higher than 5 in most of the fish examined. High ratio values were noted primarily in small specimens, and the fish inhabiting the Baltic Sea are primarily small species. From 70 to 90% of the Baltic Sea fish examined had Se:Hg molar ratios that exceeded 5, except for flounder (58%). Only in large cod (greater than 100 cm), which represented 3.8% of the cod examined, was the value of this ratio less than 1. This indicates that the commercial fish in the Baltic Sea have low concentrations of mercury and high Se:Hg values, which guarantee safe fish consumption. The Se:Hg molar ratio could be an additional criterion, along with measuring Hg concentrations, for assessing the risk of mercury exposure from Baltic Sea fish. Considerations of the potential protection of selenium against mercury toxicity might be important when assessing risk to people who consume fish frequently. It will be difficult to communicate this message based on molar ratio values since it requires knowledge of mercury and selenium concentrations in fish of different species and sizes from various regions. Threshold values must be assigned to different human populations. The current study indicates that the range of Se:Hg ratio values within species differ widely, which further complicates assigning threshold values. Therefore, in my opinion, using this ratio at present as the main criterion in mercury toxicity assessment is premature. Acknowledgments The author would like to thank Leszek Barcz and the crew of the r/v Baltica for the collection of fish samples. References Afonso, C., Lourenco, H. M., Pereira, C., Martins, M. F., Carvalho, M. L., Castro, M., et al. (2008). Total and organic mercury, selenium and a-tocopherol in some
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