0ACTA VET. BRNO 2008, 77: 637-643; doi:10.2754/avb200877040637
Mercury and Methylmercury Concentrations in Muscle Tissue of Fish Caught in Major Rivers of the Czech Republic
K. KRUZiKOVA1, t. randAk2, r. kensovA1, h. kroupovA2, d. leontovycovA3,
z. svobodovA12
11 Department of Veterinary Public Health and Toxicology, Faculty of Veterinari Hygiene and Ecology, University of Veterinary and Pharmaceutical Sciences Brno, Czech Republic 2) Research Institute of Fish Culture and Hydrobiology Vodnany, University of South Bohemia,
Czech Republic 3) Czech Hydrometeorological Institute Prague
Received February 13, 2008 Accepted June 11, 2008
Abstract
Kruzikova K., T. Randak, R. Kensova, H. Kroupova, D. Leontovycova, Z. Svobodova: 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, Ohre, Vltava, Berounka, Sazava, Otava, Luznice, 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 Obristvi, 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 Obristvi 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 Obristvi 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; Houserova 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 (Marsalek et al. 2007), with most attention paid to the longest Czech river, Labe (Dusek et al. 2005; Marsalek et al. 2006; Zlabek et al. 2005). In 2007, attention focused on important outlet sections of rivers and their variables at the
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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.
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. Luznice - Bechyne (river km 11), Otava - Topelec (river km 20), Sazava - Nespeky (river km 27.5), Berounka - Srbsko (river km 29), Vltava - Zelcin (river km 5), Labe - Obfistvi (river km 122), Ohfe - Terezin (river km 3), Labe - Decin (river km 21), Svratka - Zidlochovice (river km 23), Dyje - Pohansko (river km 16), Morava - Lanzhot (river km 9.5), Odra -Bohumin (river km 9) (Fig. 1). River kilometres of the Labe sites (Obfistvi and Decin) and Odra site (Bohumin) were measured from the border with Germany and Poland.
Fig 1. The map shows localities where fish were caught
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; Marsalek and Svobodova 2006). Samples were prepared by acidic digestion and extraction to toluene (Marsalek and Svobodova 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 x 0.53 mm x 0.83 ^m; 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 ^g 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.
Materials and Methods
Legend:
1. Luznice - Bechyne
2. Otava - Topelec
3. Sazava - Nespeky
4. Berounka - Srbsko
5. Vltava - Zelcin
6. Labe - Obfistvi
10. Dyje - Pohansko
11. Morava - Lanzhot
12. Odra - Bohumin
7. Ohfe - Terezin
8. Labe - Decin
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 Luznice - Bechyne 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.
Table 1. The main characteristic of sampled chub (Leuciscus cephalus)
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 and0.076 ± 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 - Obristvi.
THg concentrations at Obristvi (Labe) were significantly higher (P < 0.05) than those found at the sites on the Berounka, Otava, Luznice, Dyje and at the Labe site of Decin. Concentrations from the Berounka site were significantly lower (P < 0.05) also compared to the Vltava site.
Locality n Body weight (g) Age (years)
mean ± SD mean ± SD
1 Luznice - Bechyne 9 182.2 ± 105.12 3.1 ± 0.60
2 Otava - Topelec 7 93.6 ± 34.12 2.9 ± 0.38
3 Sazava - Nespeky 9 105.6 ± 44.82 2.4 ± 0.53
4 Berounka - Srbsko 9 265.0 ± 72.24 3.4 ± 0.53
5 Vltava - Zelcin 9 282.7 ± 84.41 4.1 ± 0.78
6 Labe - Obristvi 10 272.0 ± 170.20 4.0 ± 1.15
7 Ohre - Louny 9 347.8 ± 75.00 4.4 ± 0.53
8 Labe - Decin 9 343.9 ± 104.01 4.3 ± 0.71
9 Svratka - Zidlochovice 7 266.4 ± 50.47 3.4 ± 0.53
10 Dyje - Pohansko 7 257.1 ± 96.95 3.9 ± 1.21
11 Morava - Lanzhot 4 426.0 ± 381.13 4.5 ± 0.58
12 Odra - Bohumin 7 194.3 ± 52.08 3.6 ± 0.53
n: number of fish examined
Luznice Otava Sâzava Berounka Vltava Labe-Obnstvi Ohfe Labe-Dêcin Svfatka Dyje Morava Odra
Fig. 2. Content of THg in selected localities b c Groups with different alphabetic superscripts differ significantly (P < 0.05)
MeHg concentrations at Obristvi on Labe were significantly higher (P < 0.05) than those found at sites on the Luznice, Berounka and Dyje. A significant difference (P < 0.05) was also found between the Luznice 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 (Zlabek et al. 2005; Dusek et al. 2005; Marsalek et
1 25 b)
rö .2D
Fig. 3. Content of MeHg in selected localities. b- c Groups with different alphabetic superscripts differ significantly (P < 0.05)
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, respectively) were found at Obfistvi on Labe (river km 122). Dusek et al. (2005) monitored Labe between 1991 and 1996. Their data showed increased mercury concentrations (0.306 mg-kg-1 in muscle tissues of omnivorous fish) in the vicinity of the Spolana Neratovice factory (river km 122). Their finding was corroborated by Marsalek et al. (2006), who in 2004 found up to 0.933 and 0.83 mg-kg-1 muscle in chub caught upstream (river km 121) and downstream (river km 116) of the Spolana Neratovice factory, respectively. Zlabek et al. (2005) also reported higher Hg concentrations in chub caught in 2003 at Lysa nad Labem on Labe (river km 160) and at Obfistvi (river km 116) (0.9 and 1.5 mg-kg-1, respectively). Similarly to other authors, they reported different Hg concentrations at different sites along Labe, which has been corroborated by our finding of 0.118 mg-kg-1 muscle at Decin, which is 100 km farther downstream Labe than the already mentioned Obfistvi site (0.263 mg-kg-1). A comparison with the findings of Zlabek et al. (2005) shows a threefold decrease in the level of contamination of Labe at that site compared with data from 2003.
In their paper on mercury concentrations found in different species of fish in Odra, Svobodova and Hejtmanek (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 of0.09-0.154 mg-kg-1. The lowest mercury concentration was found in the river Morava that runs to Austria.
HgMe/THg ratio
Fig. 4 shows the differences in percentages of HgMe to total mercury at individual sites. To calculate HgMe/THg ratios, MeHg was expressed as Hg weight and related to THg (in %). The highest percentage of HgMe to total mercury was found at Obfistvi on Labe (90.4%), and the lowest percentage of HgMe /THg was found in muscle tissue of fish caught in Vltava and Odra (76 and 77% HgMe to THg, respectively). No significant differences were found between the monitored sites.
Houserova et al. (2006a) and Marsalek et al. (2005) also reported high percentages of HgMe to THg ranging from 74 do 100% in their chub studies. In their analysis of muscle tissue of carp (Cyprinus carpio), Marsalek et al. (2007) reported high percentages of HgMe to THg (87.9-98.5%), i.e. similar percentages of methylmercury to total mercury as those found
D Median □25»75% 110%-90%
Luänice Otava Säzava Berounka Vitava Labe-Obnstvi Ohre Labe-Decin Svratka Dyje Morava Odra
Fig. 4. Mean percentages of HgMe to total mercury in muscle tissue of fish from individual localities. Dark columns show percentage of HgMe, THg is 100%.
in our analysis of chub, which ranged from 53.6 to 99.6%. Kannan et al. (1998) also mentioned differences in HgMe/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 (Marsalek 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 Obristvi on Labe the hazard index for fisherman's family is 1.365, which indicates a possible hazard associated with fish consumption. ^u.oTTjj i 4JJ i The World Health Organisation
iable 2. Hazard indices for a standard consumer and a member of niTUTYv u u u
a fisherman's family, and maximum tolerable weekly intakes of chub (WHO) naS Deen °DserVing meat from monitored localities the MeHg issue and has set
a maximum recommended dose of MeHg, the so-called Provisional Tolerable Weekly Intake (PTWI), at 1.6 ^g MeHg-kg1 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
Hazard index for Hazard index for Maximum weekly
standard consumer* fisherman's family* tolerable intake** (kg)
Luznice-Bechyne 0.014 0.571 1.00
Otava-Topelec 0.014 0.545 1.13
Sazava-Nespeky 0.022 0.882 0.76
Berounka-Srbsko 0.013 0.503 1.27
Vltava-Zelcin 0.023 0.903 0.80
Labe-Obristvi 0.034 1.365 0.44
Ohfe-Terezin 0.016 0.664 0.99
Labe-Decin 0.015 0.612 1.06
Svratka-Zidlochovice 0.016 0.628 0.98
Dyje-Pohansko 0.013 0.503 1.47
Morava-Lanzhot 0.020 0.799 0.81
Odra-Bohumin 0.017 0.659 1.04
* calculation of THg according to Kannan (1998) ** calculation of MeHg according to WHO
week. On the other hand, the maximum amounts of fish that can be consumed per week from Labe - Obfistvi and Vltava - Zelcin 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.
Obsah rtuti a methylrtuti ve svalovine ryb z vyznamnych ceskych rek
Cilem pfedkladané prace bylo zhodnotit dvanact zavërovych profilû fek na ùzemi Ceské republiky (Labe, Ohfe, Vltava, Berounka, Sazava, Otava, Luznice, Svratka, Dyje, Morava, Odra) z hlediska jejich zatizeni rtuti. Jako indikatoru byla pouzita svalovina jelce tloustë (Leuciscus cephalus) odloveného v roce 2007 na vybranych lokalitach. Celkem bylo ana-lyzovano 96 kusû ryb. Celkova rtut' byla mëfena metodou atomové absorpcni spektrofoto-metrie na analyzatoru AMA 254 a methylrtut' metodou plynové chromatografie s detekci elektronového zachytu. Obsah celkové rtuti (THg) ve svalovinë se pohyboval v rozmezi 0,039-0,384 mg^kg-1 cerstvé tkanë a methylrtuti (MeHg) v rozmezi 0,033-0,362 mg-kg-1 cerstvé tkanë. Rtut' vazana v methyrtuti (HgMe) zaujima v prûmëru cca 82,2% z celkové rtuti. Nejvëtsi zatizeni rtuti bylo zjistëno v rybach z feky Labe v lokalitë Obfistvi (THg 0,263 ± 0,086 mg-kg-1; MeHg 0,256 ± 0,084 mg-kg-1). Ryby z fek vytékajicich za hranice CR (Labe, Odra, Morava) maji nizké hodnoty obsahu rtuti. CR se tak nepodili vyznamnou mërou na jejich dalsim znecistëni za hranicemi cr. Index rizika ze sledovanych lokalit je nëkolikanasobnë nizsi nez 1, pouze v lokalitë Labe - Obfistvi dosahuje hodnoty 1,365 pro cleny rybafské rodiny (tj. pfi konzumaci 10 kg ryb rocnë). To ukazuje na mozné riziko kon-zumace rybiho masa z dané lokality. Na zakladë hodnot PTWI methylrtuti bylo stanoveno nejvyssi mnozstvi rybiho masa, které lze zkonzumovat za tyden. Nejnizsi hodnota byla v lokalitë Labe - Obfistvi (0,44 kg). Vysledky prace jsou dilcim pfispëvkem pro hodnoceni zdravotniho rizika na nejvyznamnëjsich tocich Ceské republiky.
Acknowledgement
This study was supported by the project MSM 6215712402 (Ministry of Education, Youth and Sports of the Czech Republic), SP/2e7/229/07 and The Biomonitoring Programme of the Czech Hydrometeorological Institute and Ministry of the Environment of the Czech Republic.
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