Mercury and Methylmercury Concentrations in Muscle Tissue of Fish Caught in Major Rivers of the Czech Republic

Kružíková K., T. Randák, R. Kenšová, H. Kroupová, D. Leontovyčová, Z. Svobodová: Mercury and Methylmercury Concentrations in Muscle Tissue of Fish Caught in Major Rivers of the Czech Republic. Acta Vet Brno 2008, 77: 637-643. The aim of the study was to evaluate mercury contamination at twelve outlet sites of rivers in the Czech Republic (Labe, Ohře, Vltava, Berounka, Sázava, Otava, Lužnice, Svratka, Dyje, Morava and Odra). As an indicator, we used muscle tissue of the chub (Leuciscus cephalus) caught at selected sites in 2007. A total of 96 fish were examined. Total mercury was determined by atomic absorption spectrophotometry using the AMA 254 analyzer and methylmercury was determined by gas chromatography with electron-capture detection. Total mercury (THg) and methylmercury (MeHg) concentrations ranged 0.039–0.384 mg·kg-1 fresh weight and 0.033–0.362 mg·kg-1 fresh weight, respectively. Mercury bound in methylmercury (HgMe) made up on average about 82.2% of total mercury. The highest mercury concentrations were found in fish from Obříství, a site on Labe (THg 0.263 ± 0.086 mg·kg-1; MeHg 0.256 ± 0.084 mg·kg-1). Mercury concentrations in fish from rivers that cross the borders of the Czech Republic (Labe, Odra and Morava) were low. The Czech Republic therefore does not contribute significantly to river pollution outside its national borders. Hazard indices of the sites monitored were well below 1, and reached 1.365 only in Obříství on Labe for fisherman’s family members (i.e. in the case of annual consumption of 10 kg fish). This indicates possible hazards involved in eating meat of fish caught in that location. Based on PTWI for methylmercury, the maximum amount of fish meat allowed for consumption per week was calculated. The site with the lowest value was Obříství on Labe (0.44 kg). The results of this study present a partial contribution to health risk assessment on the major rivers in Czech Republic. Leuciscus cephalus, methylmercury/mercury ratio, THg, MeHg, hazard index, PTWI Mercury is a global pollutant and is distributed in the natural environment including bioorganisms. Among naturally and anthropogenically occurring types of mercury, methylmercury (MeHg) is the most hazardous for human health, especially for the developing foetus (Yasutake et al. 2005). As it advances in the food chain, MeHg accumulates in fish, and fish thus become the main source of human contamination with methylmercury (WHO 1990). Mercury bound in methylmercury (HgMe) makes up 70–95% of total mercury (THg) in fish tissues (Mason et al. 1995; Houserová et al. 2006b). Mercury (like PCBs and other compounds) has been shown to affect the endocrine systems and reproductive success of several fish species (Friedmann et al. 2002). The monitoring of mercury contamination of surface water and the evaluation of health hazards related to fish consumption have been given much attention in the Czech Republic (Maršálek et al. 2007), with most attention paid to the longest Czech river, Labe (Dušek et al. 2005; Maršálek et al. 2006; Žlábek et al. 2005). In 2007, attention focused on important outlet sections of rivers and their variables at the ACTA VET. BRNO 2008, 77: 637-643; doi:10.2754/avb200877040637 Address for correspondence: Ing. Kamila Kružíková University of Veterinary and Pharmaceutical Sciences Palackého 1-3 612 42 Brno Czech Republic Phone: +420 5 4156 2783 Fax: +420 5 4156 2790 E-mail: kruzikovak@vfu.cz http://www.vfu.cz/acta-vet/actavet.htm borders with our countries. That also included sites that characterize the rivers before they leave the Czech Republic and enter neighbouring states. Thus, it was possible to estimate the role of the Czech Republic in the contamination of rivers that transverse several countries. The aim of our study was to analyse the total mercury and methylmercury content in muscle of chub caught in eleven most important rivers in the Czech Republic, to assess mercury contamination contributions of individual localities and to evaluate health hazards of eating fish from the selected rivers. Materials and Methods A total of 96 indicator fish (Leuciscus cephalus males) were collected at the selected localities in 2007. The chub came from the twelve sites on eleven major rivers in the Czech Republic, i.e. Lužnice Bechyně (river km 11), Otava Topělec (river km 20), Sázava Nespeky (river km 27.5), Berounka Srbsko (river km 29), Vltava Zelčín (river km 5), Labe Obříství (river km 122), Ohře Terezín (river km 3), Labe Děčín (river km 21), Svratka Židlochovice (river km 23), Dyje Pohansko (river km 16), Morava Lanžhot (river km 9.5), Odra Bohumín (river km 9) (Fig. 1). River kilometres of the Labe sites (Obříství and Děčín) and Odra site (Bohumín) were measured from the border with Germany and Poland. The fish were collected after they were stunned with electrofishing equipment. They were immediately weighed and muscle tissue samples were taken for analysis of total mercury and methylmercury. The age of fish was determined from their scales. The sex of fish was determined macroscopically. Samples of muscle tissue were put into polyethylene bags, labelled and stored in a freezer at -18 °C. Fish muscle mercury (THg) concentrations were determined by cold vapour atomic absorption spectrometry on AMA 254 (Altec Ltd., Czech Republic) analyser. Methylmercury (MeHg) was determined in the form of methylmercury chloride by gas chromatography (Caricchia et al. 1997; Maršálek and Svobodová 2006). Samples were prepared by acidic digestion and extraction to toluene (Maršálek and Svobodová 2006). Shimadzu capillary gas chromatograph with an electron captured detector GC 2010A (Shimadzu Kyoto, Japan) was used for the analysis. The capillary column DB 608 (30 m × 0.53 mm × 0.83 μm; J&W Scientific Chromservis, Czech Republic) was used. Evaluation was made using GC Solution software (Shimadzu Kyoto, Japan) and MS Excel software. The limit of detection (LOD) was set as a sum of triple the standard deviation of a blank and a blank mean value. Detection limit for THg and MeHg determination methods were 1 mg·kg-1 and 21 mg·kg-1, respectively. Accuracy of the results of THg and MeHg was validated using standard reference material BCR-CRM 464 (Tuna Fish, IRMM, Belgium). Total mercury and methylmercury concentrations are given in mg·kg-1 fresh weight (FW). For the methylmercury/ mercury ratio evaluations, the methylmercury value was expressed as HgMe. The hazard index was calculated according to Kannan et al. (1998) using a reference dose (RfD) for THg (0.3 mg·kg-1 body weight per day) set forth by US EPA. To determine the maximum possible consumption of fish meat, the provisional tolerable weekly intake limit (PTWI) of 1.6 μg MeHg per kg body weight per week was used (WHO 1990). Statistical analysis of the data was performed using the program STATISTICA 8.0 for Windows (StatSoft CR). The data were assessed by non-parametric Kruskal-Wallis test because data normality was not proven. Whenever the Kruskal-Wallis test showed significant differences between profiles (P < 0.05), multiple comparisons of all profiles were subsequently performed. 638 Fig 1. The map shows localities where fish were caught Legend: 1. Lužnice Bechyně 7. Ohře Terezín 2. Otava Topělec 8. Labe Děčín 3. Sázava Nespeky 9. Svratka Židlochovice 4. Berounka Srbsko 10. Dyje Pohansko 5. Vltava Zelčín 11. Morava Lanžhot 6. Labe Obříství 12. Odra Bohumín Results and Discussion Total mercury and methylmercury concentrations in fish Total mercury and methylmercury was found in all 96 examined samples, but in five samples from Lužnice Bechyně and one sample from Berounka Srbsko, methylmercury concentrations were below the detection limit. The main characteristics of the caught fish are given in Table 1. Medians of total mercury and methylmercury concentrations from individual sites are given in Figs 2 and 3. The lowest concentrations of mercury and methylmercury were 0.097 ± 0.032 mg·kg-1 and 0.076 ± 0.035 mg·kg-1, respectively. These concentrations were found in chub from Dyje Pohansko. On the other hand, the highest concentrations of total mercury and methylmercury (0.263 ± 0.086 mg·kg-1 and 0.256 ± 0.084 mg·kg-1, respectively) were found in chub from Labe Obříství. THg concentrations at Obříství (Labe) were significantly higher (P < 0.05) than those found at the sites on the Berounka, Otava, Lužnice, Dyje and at the Labe site of Děčín. Concentrations from the Berounka site were significantly lower (P < 0.05) also compared to the Vltava site. MeHg concentrations at Obříství on Labe were significantly higher (P < 0.05) than those found at sites on the Lužnice, Berounka and Dyje. A significant difference (P < 0.05) was also found between the Lužnice and the Vltava sites. Long-term high mercury contamination of the aqueous environment has been observed in Labe. Contamination monitoring there has been going on for many years, and has been documented in a number of studies (Žlábek et al. 2005; Dušek et al. 2005; Maršálek et 639 Table 1. The main characteristic of sampled chub (Leuciscus cephalus) n: number of fish examined Locality Body weight (g) Age (years) n mean ± SD mean ± SD 1 Lužnice Bechyně 9 182.2 ± 105.12 3.1 ± 0.60 2 Otava Topělec 7 93.6 ± 34.12 2.9 ± 0.38 3 Sázava Nespeky 9 105.6 ± 44.82 2.4 ± 0.53 4 Berounka Srbsko 9 265.0 ± 72.24 3.4 ± 0.53 5 Vltava Zelčín 9 282.7 ± 84.41 4.1 ± 0.78 6 Labe Obříství 10 272.0 ± 170.20 4.0 ± 1.15 7 Ohře Louny 9 347.8 ± 75.00 4.4 ± 0.53 8 Labe Děčín 9 343.9 ± 104.01 4.3 ± 0.71 9 Svratka Židlochovice 7 266.4 ± 50.47 3.4 ± 0.53 10 Dyje Pohansko 7 257.1 ± 96.95 3.9 ± 1.21 11 Morava Lanžhot 4 426.0 ± 381.13 4.5 ± 0.58 12 Odra Bohumín 7 194.3 ± 52.08 3.6 ± 0.53 Fig. 2. Content of THg in selected localities a, b, c, Groups with different alphabetic superscripts differ significantly (P < 0.05) TH g (m g· kg -1 ) al. 2006). Of all the sites monitored in the present study, the highest concentrations of Hg and MeHg (0.263 mg·kg-1 and 0.256 mg·kg-1,

Mercury is a global pollutant and is distributed in the natural environment including bioorganisms.Among naturally and anthropogenically occurring types of mercury, methylmercury (MeHg) is the most hazardous for human health, especially for the developing foetus (Yasutake et al. 2005).As it advances in the food chain, MeHg accumulates in fish, and fish thus become the main source of human contamination with methylmercury (WHO 1990).Mercury bound in methylmercury (Hg Me ) makes up 70-95% of total mercury (THg) in fish tissues (Mason et al. 1995;Houserová et al. 2006b).Mercury (like PCBs and other compounds) has been shown to affect the endocrine systems and reproductive success of several fish species (Friedmann et al. 2002).
The monitoring of mercury contamination of surface water and the evaluation of health hazards related to fish consumption have been given much attention in the Czech Republic (Maršálek et al. 2007), with most attention paid to the longest Czech river, Labe (Dušek et al. 2005;Maršálek et al. 2006;Žlábek et al. 2005).
In 2007, attention focused on important outlet sections of rivers and their variables at the borders with our countries.That also included sites that characterize the rivers before they leave the Czech Republic and enter neighbouring states.Thus, it was possible to estimate the role of the Czech Republic in the contamination of rivers that transverse several countries.
The aim of our study was to analyse the total mercury and methylmercury content in muscle of chub caught in eleven most important rivers in the Czech Republic, to assess mercury contamination contributions of individual localities and to evaluate health hazards of eating fish from the selected rivers.
The fish were collected after they were stunned with electrofishing equipment.They were immediately weighed and muscle tissue samples were taken for analysis of total mercury and methylmercury.The age of fish was determined from their scales.The sex of fish was determined macroscopically.Samples of muscle tissue were put into polyethylene bags, labelled and stored in a freezer at -18 °C.
Fish muscle mercury (THg) concentrations were determined by cold vapour atomic absorption spectrometry on AMA 254 (Altec Ltd., Czech Republic) analyser.
Methylmercury (MeHg) was determined in the form of methylmercury chloride by gas chromatography (Caricchia et al. 1997;Maršálek and Svobodová 2006).Samples were prepared by acidic digestion and extraction to toluene (Maršálek and Svobodová 2006).Shimadzu capillary gas chromatograph with an electron captured detector GC 2010A (Shimadzu Kyoto, Japan) was used for the analysis.The capillary column DB 608 (30 m × 0.53 mm × 0.83 μm; J&W Scientific Chromservis, Czech Republic) was used.Evaluation was made using GC Solution software (Shimadzu Kyoto, Japan) and MS Excel software.
The limit of detection (LOD) was set as a sum of triple the standard deviation of a blank and a blank mean value.Detection limit for THg and MeHg determination methods were 1 mg•kg -1 and 21 mg•kg -1 , respectively.Accuracy of the results of THg and MeHg was validated using standard reference material BCR-CRM 464 (Tuna Fish, IRMM, Belgium).
Total mercury and methylmercury concentrations are given in mg•kg -1 fresh weight (FW).For the methylmercury/ mercury ratio evaluations, the methylmercury value was expressed as Hg Me .
The hazard index was calculated according to Kannan et al. (1998) using a reference dose (RfD) for THg (0.3 mg•kg -1 body weight per day) set forth by US EPA.
To determine the maximum possible consumption of fish meat, the provisional tolerable weekly intake limit (PTWI) of 1.6 μg MeHg per kg body weight per week was used (WHO 1990).
Statistical analysis of the data was performed using the program STATISTICA 8.0 for Windows (StatSoft CR).The data were assessed by non-parametric Kruskal-Wallis test because data normality was not proven.Whenever the Kruskal-Wallis test showed significant differences between profiles (P < 0.05), multiple comparisons of all profiles were subsequently performed.

Total mercury and methylmercury concentrations in fish
Total mercury and methylmercury was found in all 96 examined samples, but in five samples from Lužnice -Bechyně and one sample from Berounka -Srbsko, methylmercury concentrations were below the detection limit.The main characteristics of the caught fish are given in Table 1.
Medians of total mercury and methylmercury concentrations from individual sites are given in Figs 2 and 3.The lowest concentrations of mercury and methylmercury were 0.097 ± 0.032 mg•kg -1 and 0.076 ± 0.035 mg•kg -1 , respectively.These concentrations were found in chub from Dyje -Pohansko.On the other hand, the highest concentrations of total mercury and methylmercury (0.263 ± 0.086 mg•kg -1 and 0.256 ± 0.084 mg•kg -1 , respectively) were found in chub from Labe -Obříství.THg concentrations at Obříství (Labe) were significantly higher (P < 0.05) than those found at the sites on the Berounka, Otava, Lužnice, Dyje and at the Labe site of Děčín.Concentrations from the Berounka site were significantly lower (P < 0.05) also compared to the Vltava site.
MeHg concentrations at Obříství on Labe were significantly higher (P < 0.05) than those found at sites on the Lužnice, Berounka and Dyje.A significant difference (P < 0.05) was also found between the Lužnice and the Vltava sites.
Long-term high mercury contamination of the aqueous environment has been observed in Labe.Contamination monitoring there has been going on for many years, and has been documented in a number of studies (Žlábek et al. 2005;Dušek et al. 2005;Maršálek et   In their paper on mercury concentrations found in different species of fish in Odra, Svobodová and Hejtmánek (1976) reported mercury concentration of about 0.18 mg•kg -1 in chub muscle tissue, which is similar to mercury concentration values found in our study (0.128 mg•kg -1 ) in 2007.
Mean mercury concentrations in muscle tissue of chub caught in rivers that run out of the Czech Republic were in the range of 0.09-0.154mg•kg -1 .The lowest mercury concentration was found in the river Morava that runs to Austria.Hg Me /THg ratio Fig. 4 shows the differences in percentages of Hg Me to total mercury at individual sites.To calculate Hg Me /THg ratios, MeHg was expressed as Hg weight and related to THg (in %).The highest percentage of Hg Me to total mercury was found at Obříství on Labe (90.4%), and the lowest percentage of Hg Me /THg was found in muscle tissue of fish caught in Vltava and Odra (76 and 77% Hg Me to THg, respectively).No significant differences were found between the monitored sites.Houserová et al. (2006a) and Maršálek et al. (2005) also reported high percentages of Hg Me to THg ranging from 74 do 100% in their chub studies.In their analysis of muscle tissue of carp (Cyprinus carpio), Maršálek et al. (2007) reported high percentages of Hg Me to THg (87.9-98.5%),i.e. similar percentages of methylmercury to total mercury as those found ) in our analysis of chub, which ranged from 53.6 to 99.6%.Kannan et al. (1998) also mentioned differences in Hg Me /THg ratios.Such differences suggest different conditions for mercury methylation in river sediment, e.g.redox conditions, types of sediment, microflora, and season of the year (Maršálek 2006).

Health hazard assessment
Potential health hazard caused by mercury in fish was calculated according to the method of Kannan et al. (1998), who described the calculation of a hazard index associated with fish consumption.Hazard index below 1 indicates no hazard for consumers.In hazard index calculations, average consumption of freshwater fish in the Czech Republic was used, i.e. 1 kg per capita (Ministry of Agriculture 2007) and 10 kg per member of fisherman's household.Hazard indices calculated for Hg are given in Table 2.The indices given are low, in fact several times lower than the hazard index of 1, only at Obříství on Labe the hazard index for fisherman's family is 1.365, which indicates a possible hazard associated with fish consumption.
The World Health Organisation (WHO) has been observing the MeHg issue and has set a maximum recommended dose of MeHg, the so-called Provisional Tolerable Weekly Intake (PTWI), at 1.6 μg MeHg•kg -1 body weight/week.This value can then be used to calculate the amount of fish meat that a consumer from a specific site can eat.From the MeHg contamination point of view, the best rivers of the ones monitored in the present study were Dyje -Pohansko and Berounka -Srbsko, where up to 1.47 kg and 1.27 kg, respectively, of fish captured can be consumed per  week.On the other hand, the maximum amounts of fish that can be consumed per week from Labe -Obříství and Vltava -Zelčín are only 0.44 kg and 0.80 kg, respectively.The limit of 0.5 mg Hg•kg -1 wet weight provided by the Commission Regulation (EC) No. 1881/2006 setting the maximum level for certain contaminants in foodstuffs, has not been exceeded in any samples of chub muscle.

638Fig
Fig 1.The map shows localities where fish were caught

Fig. 2 .
Fig. 2. Content of THg in selected localities a, b, c, Groups with different alphabetic superscripts differ significantly (P < 0.05)

Fig. 4 .
Fig. 4. Mean percentages of Hg Me to total mercury in muscle tissue of fish from individual localities.Dark columns show percentage of Hg Me , THg is 100%.