Slov Vet Res 2005; 42 (1/2): 15-21 UDC 614.31:615.9:543.06:639.3 Original Research Paper THE CONTENTS OF Cu, Zn, Fe AND Mn IN SLOVENIAN FRESHWATER FISH Zlatka Bajc1*, Ksenija Sinigoj Gacnik1, Vlasta Jencic2, Darinka Z. Doganoc1 Addresses of authors: 1 Institute for Food Hygiene and Bromatology, 2 Institute for Breeding and Health Care of Wild Animals Fishes and Bees, Veterinary Faculty, Gerbiceva 60, 1000 Ljubljana, Slovenia * Chorresponding avtor, E-mail: zlatka.bajc@vf.uni-lj.si Summary: From 1999 to 2003, 141 samples of freshwater fish from various Slovenian rivers and brooks were analysed for their content of Cu, Zn, Fe and Mn. The concentrations of elements were determined in the muscle/skin (in natural proportion), head, liver and kidney. Brown trout (Salmo trutta m. fario), marble trout (Salmo marmoratus), brook trout (Salevelinus fontinalis), rainbow trout (Oncorhynchus mykiss), grayling (Thymallus thymallus), chub (Leuciscus c. cephalus), nase (Chondrostoma nasus), Danube roach (Rutilus pigus virgo) and barbel (Barbus barbus) were examined. Flame atomic absorption spectrometry was used. The distribution of trace elements in various tissues was studied. It was found that the distribution is specific for each element and also varied by fish species. The contents of elements were the lowest in fish muscle for all species. The target tissues for the elements examined are the metabolically active ones, such as the liver and kidney. Key words: food contamination; food analysis; copper; zinc; iron; manganese; spectrometry; fishes Introduction Fish represent a high quality source of dietary protein, but could lose these properties due to environmental contamination. Contaminants can be introduced into the environment through natural sources, industry, urbanisation and intensive agriculture. In aquatic ecosystems, heavy metals have received considerable attention due to their toxicity and accumulation. Some metals are toxic for living organisms even at low concentrations. Others are essential and necessary in trace amounts for the functioning of biological systems but can also be toxic at higher concentrations (1, 2). Aquatic organisms, especially fish located at the end of the aquatic food chain, are widely used for biological monitoring variation of environmental levels of anthropogenic pollutants (1, 2, 3, 4, 5). Fish receive trace elements, either directly from the water through their grills, or indirectly from food through the alimentary tract (2). Together with biological factors such as feeding behaviour and interactions between elements, chemical factors such as acidity, buffer capacity, and the presence of calcium and organic compounds in water may influ- Received: 22 April 2005 Accepted for publication: 9 September 2005 ence the bioavailability and accumulation of heavy metals in fish (2, 6, 7). The aim of this study was to determine the content of Cu, Zn, Fe and Mn in the muscle/skin in natural proportion, heads, livers and kidneys of freshwater fish caught in Slovenian rivers and brooks. The distribution of elements in the fish body and the differences in level of accumulation in various fish species were investigated. Material and methods Samples From 1999 to 2003, 141 freshwater fish samples caught in various Slovenian rivers and brooks were analysed. The fish were sampled by local fisherman and sent to the laboratory either fresh or frozen. Samples were sorted by date and location of catch, fish species, length and weight. 74 samples of various species of salmonids such as brown trout (Salmo trutta m. fario), marble trout (Salmo marmoratus), brook trout (Salevelinus fontinalis) and rainbow trout (Oncorhynchus mykiss), 23 samples of chub (Leuciscus c. cephalus), 12 samples of nase (Chondrostoma nasus), 9 samples of Danube roach (Rutilus pigus virgo), 12 samples of grayling 16 Z. Bajc, K. Šinigoj Gačnik, V. Jenčič, D. Z. Doganoc Table 1: Fish species, rivers and number of samples analysed including the Debenski potok and Titanova mlinščica brooks 1 including the Lepena and Doblarec brooks (Thymallus thymallus) and 11 samples of barbel (Barbus barbus) were examined in the study. The number of samples, fish species and location of catch (name of open water) are listed in Table 1. The rivers and brooks in Table 1 are sorted in three groups (I, II, III). Group I includes the Sava River with subsidiary streams and Group II includes the Drava River with subsidiary streams. The rivers and brooks in the first two groups flow to the Black Sea. In Group III are rivers and brooks that flow to the Adriatic Sea. From 1 to 3 fish were prepared per sample, according to the procedure described in the Association of Official Analytical Chemists (8). Before preparation the fish were washed, dried and scaled. For each fish, the head, liver and kidney were sampled and fillets in natural proportion were prepared from muscle and skin. The fillets were then minced with a Buchi-400 homogenizer. Different tissues were packed separately and kept below -18° C until the day of analysis. Some of the fish samples received were already dissected and without vis- cera and/or heads, and in those cases only the muscle/skin was prepared. In some cases only the liver and kidney were received, but for all of them the data regarding species, weight, length, date and location of catch were available. Reagents Standard solutions of Cu, Zn, Fe and Mn were prepared from commercial stock standard solutions (Merck) at concentrations of 1000 mg/L. Double deionised water was used throughout. Working standard solutions were prepared by dilution of stock standard solution with the addition of hydrochloric acid, so that the acid concentration in working standard solutions matched the acid concentration in digested solutions. All reagents used were an analytical-reagent grade. Sample preparation 10 g of muscle/skin or whole head, liver or kidney were weighed in quartz-glass crucibles, dried The contents of Cu, Zn, Fe and Mn in Slovenian freshwater fish 17 Table 2: Results of analyses of certified reference materials. Mean and standard deviation in mg/kg. Number of analysis in parentheses Metal BCIi NU185R BCR ^422 BC R rV'ISG Cu Found 0,533 ±0.021 (6) 0.84 ±0.06 (4) 28,8 ± ! .2 (5) Certified 0.544 ±0.017 1.05 ±0.07 31,9± 0.4 Zn Kound 128.6 ±2.6 (6) 19,2 ±0.4 (6) 119 ±7 (6) Certified 138,6± 2.1 19,6 ±0.5 128 ±6 Fe Found t -1.66 ± 0.59 (5) 278 ± 17(6) Certified i 5.46 ± 0.30 299 ± 10 Mn Found 10.66 ± 0.2-1 (6) 0.512:1: 0.023 (6) 8.0 ±0.5 (6) Certified 11.07 ±0.29 0.543 ± 0.028 8.5 ± 0.3 in a drying oven at 105° C and ashed overnight at 450° C ± 25° C in a programmable furnace. The ash was dissolved with diluted hydrochloric acid (1/1, v/v). The solution was evaporated to dryness. The remains were redissolved with diluted hydrochloric acid (1/10, v/v), filtered into a 50 mL volumetric flask and diluted with water to the mark. Sample analysis The Cu, Zn, Fe and Mn concentrations in the samples were determined by flame atomic absorption spectrophotometry (FAAS), using a Varian SpectrAA 220 instrument with deuterium background correction. The determinations were preformed by aspirating the final solutions into an AA spectrophotometer using an air-acetylene flame. Absorption wavelengths were as follows: 324.8 nm for Cu, 213.9 nm for Zn, 248.3 nm for Fe and 279.5 nm for Mn. Concentrations of metals in samples were evaluated using an external standard method. The accuracy of the analytical procedure was checked by analyzing three different standard reference materials: cod muscle BCR 422, bovine liver BCR 185R and pig kidney BCR 186. The agreement for all the elements investigated was 80-98%. The results of analysis of standard reference material are shown in Table 2. A t-test was used to statistically evaluate the sample data. A p value lower than 0.05 was considered to be significant. If the concentration of a metal was below the detection limit, a value of half of the detection limits was used in order to facilitate statistical comparisons Results The contents of the metals examined in various tissues of various fish species are given in Table 3. The data in Table 3 reveal low levels of Cu and Mn in tissues and higher levels of Zn and Fe. The lowest concentrations of all elements were found in muscle/skin. The average concentrations of Cu in tissues of various fish species are shown in Fig. 1. The highest concentrations of Cu were always found in the liver, followed by the kidney, head and muscle/skin. The ratio between the level of Cu in the liver and muscle/skin was particularly high in salmonids. The concentration of Cu in the liver of other fish species was approximately ten times lover than in salmonids and we did not observe any significant differences between them. The kidney, head and muscle/skin of barbel contained significantly higher levels of Cu than chub, nase, Danube roach and grayling. The kidney and muscle/skin, but not the head, of salmonids also contained significantly higher concentrations of Cu than the species mentioned above. The average concentrations of Zn in the tissues of various fish species are shown in Figure 2. Chub and Danube roach contained the highest amount of Zn in the kidney, and nase, grayling and barbel in the head. Salmonids contained an equal amount, and the highest amount of Zn in the head and liver. The kidney of barbel, nase, salmonids and grayling contained on average less than 25 mg Zn/kg. Higher concentrations were detected in the kidney of chub and the highest in the kidney of Danube roach. In the liver, concentrations of Zn were significantly higher in Danuble roach and chub than in barbel, nase and grayling. In the muscle/skin, the highest concentrations were also detected in Danube roach and the lowest in barbel and grayling. In the head, higher concentrations of Zn were detected in chub, nase and Danube roach than in salmonids, grayling and barbel. Figure 3 presents the average concentrations of Fe in the tissues of various fish species, from 18 Z. Bajc, K. Šinigoj Gačnik, V. Jenčič, D. Z. 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