ࡱ> ` fbjbj ;kh& r r r 8 !< !(!" " " "" # #lnnnnnn$}h)="")=)= " "cOcOcO)=x  " "lcO)=lcOcOBRR "! @Ur JZRT+0bR,sqNsRR"sR#+:cO2 7### OX###)=)=)=)=   d       BIOMONITORING OF HEAVY METALS IN FISH FROM THE DANUBE RIVER Snje~ana Zrn i1, Dra~en Orai1, Marko aleta2, }eljko Mihaljevi1, Davor Zanella2 and Nina Biland~i1 1Croatian Veterinary Institute, Savska 143, Zagreb, Croatia 2 Faculty of Science, Department of Zoology, Rooseveltov trg 6, Zagreb, Croatia Corresponding author:  HYPERLINK "mailto:zrncic@irb.hr" zrncic@irb.hr Phone: +385 1 6123 663 Fax: + 385 1 6190 841 Abstract The Croatian part of the Danube River extends over 188 km and comprises 58% of the countrys overall area used for commercial freshwater fishing. To date, the heavy metal contamination of fish in the Croatian part of the Danube has not been studied. The main purpose of this study was to determine heavy metal levels in muscle tissue of sampled fish species and to analyze the measured values according to feeding habits of particular groups. Lead ranged from 0.015 g-1 dry wt in planktivorous to 0.039 g-1 dry wt in herbivorous fish, cadmium from 0.013 g-1 dry wt in herbivorous to 0.018 g-1 dry wt in piscivorous fish, mercury from 0.191 g-1 dry wt in omnivorous to 0.441 g-1 dry wt in planktivorous fish and arsenic from 0.018 g-1 dry wt in planktivorous to 0.039 g-1 dry wt in omnivorous fish. Among the analysed metals in muscle tissue of sampled fish, only mercury exceeded the maximal level (0.5 mg kg-1) permitted according to the national and EU regulations determining maximum levels for certain contaminants in foodstuffs, indicating a hazard for consumers of fish from the Danube River. Key words: Heavy metals, Danube River, Freshwater fish, Mercury Introduction Globally, freshwater fish are the most heavily exploited aquatic resources, representing about a quarter (some 20 million tons per year) of the worlds food from water (FAO 2008). Aquaculture production in European countries is decreasing due to low production efficiency. The decrease in aquaculture production has inevitably led to an increase in fishing pressures on freshwater fishery resources because of increasing market demand for caught fish. A considerable portion of the catch comes from recreational fisheries, which increases the access of inhabitants to fish from inland waters and leads to increased exposure to all treats from insufficiently controlled open waters. Fish from open waters are considered wild animals as there is no possibility to control the composition of their growing environment (Clarkson 1995). Those fish as part of the aquatic food chain are the most likely route of human exposure to contaminants. The frequency and intensity of water contamination by heavy metals depends mainly on human activities, and their levels increase due to urbanization, industry, agriculture or mining. Sometimes, contamination of the aquatic environment can be of geological origin, i.e. from ore-bearing rocks, forest fires or vegetation (Fernandez and Olalla 2000). Heavy metals may enter the fish either through direct consumption of water or organisms or by uptake through epithelia like the gills, skin and digestive tract (Burger et al. 2002). A third possible route is via sediment (Bervoets et al. 2001) which constitutes the most important reservoir of metals and other toxicants in aquatic environments. Fish feeding on benthic organisms are directly exposed to contaminated sediments while others are exposed when toxicants from sediments are resuspended into the water column. In the aquatic environment, metal toxicity can be influenced by various abiotic factors such as oxygen, hardness, pH, alkalinity or water temperature (Ghillebaert et al. 1995; Adhikari et al. 2006). The Danube River, Europes second longest river (2,857 km) flows through nine European countries. The Croatian part of the Danube is 188 km long and forms part of Croatias eastern border with Serbia. The Danube catchments area in Croatia comprises 32,800 km2 or 58% of the countrys overall area and it is widely used for commercial freshwater fishing. To date, heavy metal contamination in the Croatian part of the Danube has not been studied, while there are some results of heavy metal research in the Danube on fish caught in Germany, Austria and Hungary (Wachs 2000): in mud, water, algae and fish in the vicinity of Vienna (Rehwold et al. 1975); in freshwater mussels in the Vienna area (Gundacker 2000); in sediment in the Hungarian part of the river (Gruiz et al. 1998); in suspended solids and sediments along the entire river flow (Woitke et al. 2003); in suspended solids, sediments and mussels along the whole river (ICPDR 2002); in sediment and aquatic plants in the Serbian part of the Danube (Pajevi et al. 2008); in liver, gills and skin of starlet at one sampling point in Hungary and two in Serbia (Poleksic et al. 2010), and in the same organs of Pontic shad from the Danube River in Serbia (Vianji-Jefti et al. 2010). Studies have also been conducted on the aquatic environment, aquatic vegetation, macroinvertebrates and fishes from the Danube delta in Romania (Andreji and Stranai 2004; Tudor et al. 2006; Diaconescu et al. 2008). Traditionally, commercial and sport fisheries are carried out in the Croatian part of the Danube River and local inhabitants usually consume the caught fish. Because of the highly developed industrial areas and intensive agricultural production upstream and adjacent to the river flow in Croatia, the main goal of the study was to determine heavy metal levels in sampled specimens. The muscle tissues of several fish species collected at three sampling points along the Danube coast were analyzed with the aim of assessing the possible health risk for humans. Another objective was to compare the level of contamination in the muscle tissue of sampled specimens depending on feeding habits. Material and methods Sampling points The study was performed at three points situated between where the Danube River enters Croatia at the Hungarian border and where it leaves Croatia at the Serbian border, in the period from April to October. The first sampling point (Fig. 1) was on the border with Hungary where the Danube River enters onto Croatian territory, at the 1436 river km (rkm) from the river source (N 455107.8, E 185115.9). The river here is 420 meters wide. The second sampling point was situated 2 km downstream of the confluence of the Drava River with the Danube River, near the village Aljmaa, at 1478 rkm (N 453153.4, E 0185716.3). The river here is 650 meters wide and the influence of the Drava River on the Danube was also investigated. Third sampling point was at 1503 rkm near Dalj (N 452857.9, E 0185920.0), where the river is 540 m wide and near the point where the Danube River leaves the Croatian territory. Samples All samples were collected only on the right bank, approximately 2 m above the water surface by means of electrofishing with a 10 kW generator. Samples for physical and chemical parameters of the water at each sampling point were determined at the time of collection of the fish. Determination of the species was performed based on coloration patterns and other morphological features using determination keys according to Vukovi & Ivanovi (1971), Pov~ & Sket (1990) and Maitland (2000). Total lengths of each fish from the particular sample were measured to the nearest mm with an ichthyometer (WILDCO Fish Measuring Board, Ben Meadows, USA) and weighed to the nearest 0.1 g using a digital scale (OHAUS Scout pro, USA). Fish for metal contamination analysis were stored in the refrigerator at 4C on the spot and transferred in cooling boxes to the laboratory where they were stored at -18C until analysis. Fish were skinned, filleted and eviscerated. A commercial grade food grinder with stainless steel cutting blades (7011HS, model HGB2WTS3, Waring Commercial, USA) was used to macerate samples. The ground tissue was then homogeneously mixed before being sent through the food grinder a second time. A subsample of the ground tissue was then collected for analysis. Reagents All reagents were of analytical reagent grade, HNO3, H2O2, and HCl (Analytical Grade, Kemika, Croatia). Double deionised water (Milli-Q Millipore, 18.2 M(cm-1 resistivity) was used for all dilutions. All plastic and glassware were cleaned by soaking in diluted HNO3 (1/9, v/v) and were rinsed with distilled water prior to use. Calibrations were prepared with element standard solutions of 1000 mg l-1 of each element supplied by Perkin Elmer. Stock solution was diluted in HNO3 (0.2%). As matrix modifiers in each atomization for Pb and Cd, 0.005 mg Pd(NO3)2 and 0.003 mgMg(NO3)2 (Perkin Elmer, USA) were used. The hydride technique for mercury determinations involves the reaction of acidified aqueous samples (3% v/v HCl) with a reducing agent 0.2% sodium borohydride in 0.05% NaOH. Determination of trace metal concentrations Samples (2g) were digested with 5ml of HNO3 (65% v/v), 1ml of H2O2 (30% v/v) with a microwave closed system Multiwave 3000 (Anton Paar, Germany). A blank digest was carried out in the same way. The program for the digestion began at a potency of 1200W then ramped for 10min, after which samples were held for 10min at 1200W. The second step began at a potency of 0 W and held for 15 min. Digested samples were diluted to a final volume of 50ml with double deionised water. All metal concentrations were determined on wet weight basis as mg kg-1. Analyses of As, Cd and Pb were done by graphite furnace-atomic absorption spectroscopy using an Analyst 800 atomic absorption spectrometer (Perkin Elmer, USA) equipped with an AS 800 auto sampler (Perkin Elmer, USA). For graphite furnace measurements, argon was used as the inert gas. Pyrolytic-coated graphite tubes with a platform were used. The atomic absorption signal was measured in peak area mode against a calibration curve. Mercury was analyzed by the cold vapour technique with a flow injection system coupled to a FIAS-100 atomic absorption spectrophotometer (Perkin Elmer, USA) equipped with an AS 93 plus auto sampler (Perkin Elmer). The limits of detection (LODs, mg kg-1) of elements were found to be: As 0.01, Cd 0.0004, Cu 0.0005, Hg 0.0004 and Pb 0.005. All specimens were run in batches that included blanks, a standard calibration curve, two spiked specimens, and one duplicate. In order to validate the method for accuracy and precision, dogfish muscle (DORM-2, National Research Council, Canada) was analyzed (n=5) as a certified reference material and the recovery (mean % recoveryS.E.) was analyzed (n=5). The recovery was 93.36.3% for As, 95.45.5% for Cd, 98.74.6% for Cu, 98.13.6% for Hg and 104.64.9% for Pb. Statistical analysis In order to the assess differences in metal concentrations in muscle tissue in different fish species according to feeding habits, ANOVA with post hoc test analyses based on the Tukey test was performed. When the ANOVA assumptions were not met (data normality and homoscedasticity), the non-parametric Kruscal-Wallis test was used. The relationship between fish size (total length, weight) and metal concentration in muscle tissue was investigated by univariate and multivariate regression analysis. Statistical analyses were performed using Stata 10.0 (StataCopr. 2005. Stata Statistical Software: Release 10.0, College Station, TX). A level of p< 0.05 was considered significant. Results and discussion A total of 201 fish were chosen for heavy metal concentration analysis from the three sampling points in May, July and September. Sampled specimens were represented by 14 different fish species that varied in total length from 83 to 860 mm and total weight from 6.3 to 7000 g. Collected fish belonged to four groups according to feeding habits (Table 1.) The water quality parameters, shown in Table 2, matched the requirements for water quality category I according to Croatian Water classification regulations (Offical Gazette 77/1998; Offical Gazette 137/2008) excluding the measured value of O2 saturation in a single sample at Batina and the concentration of nitrates at the sampling site Dalj. Considering these values, it is not possible to correlate the measured metal concentrations with water properties as observed in Bervoets et al. (2001), defined the influence of pH on bioavailability of metals and increased bioaccumulation of Hg. But it is widely known that temporal oxygen deficiency enhances mobility of Hg in freshwater ecosystems (Boening 2000) and therefore it is not possible to neglect the aberration of O2 saturation even in a single measurement. The results did not indicate any differences between the measured metal concentrations from the three sampling points, which are situated very close to one another. Statistical analysis showed that the differences between heavy metal concentrations by sampling site and time of sampling were not significant (p>0.05). Metal concentrations and the corresponding mean standard deviations (expressed as g -1 dry wt) measured in muscle tissues are reported in Table 1 for each fish species. Lead ranged from 0.015 g-1 dry wt in planktivorous to 0.039 g-1 dry wt in herbivorous fish, cadmium from 0.013 g-1 dry wt in herbivorous to 0.018 g-1 dry wt in piscivorous fish, mercury from 0.191 g-1 dry wt in omnivorous to 0.441 g-1 dry wt in planktivorous fish and arsenic from 0.018 g-1 dry wt in planktivorous to 0.039 g-1 dry wt in omnivorous fish (Table 3). Among the four metals analysed in muscle tissue of sampled fish, only mercury exceeded the maximum level (0.5 mg kg-1) permitted according to national (Official Gazette 154/2008) and EU regulations (2006) determining the maximum levels for certain contaminants in foodstuffs. This corresponds to the results of Diaconescu et al. (2008) for analysed muscle tissues of mackerel and bream sampled at different sites along the Danube River in Romania. The differences between the mean concentration of measured values in the four groups according to feeding habits were significant (p<0.001). The highest concentrations of mercury were expected in the muscle tissue of piscivorous fish as described by Havelkova et al. (2008), where predatory fish had much higher concentrations than non-predatory due to their position in the food chain. In the present study, the highest mean concentration of mercury was measured in the muscle tissue of planktivorous fish, which could be explained by the body size of the collected specimens, with body lengths ranging from 780 to 860 mm and weighing 5100 to 7000 grams. These results are supported by the findings of Jackson (1991) who indicated that bioaccumulation of mercury is very size dependant. The highest concentration of mercury in an individual specimen was detected in the muscle tissue of two specimens of asp (Aspius aspius; 0.716 and 0.79 g/g dry wt.) belonging to piscivorous fish, and one specimen of grass carp (Ctenopharyngodon idella; 0.937 g/g dry wt), belonging to herbivorous fish (Table 1, Fig. 2). All sampled specimens were analyzed in respect to the time of sampling and the sampling site (variables month and sampling point) in the multivariate regression analysis. There was a significant increase in mercury concentration with an increase of fish total length (R=0.0042 p<0.001) and fish weight (R=0.0000516, p<0.001), which corroborates previous findings, such as Lange et al. (1993), who observed that fish age was more correlated with mercury concentration than fish size, and Duek et al. (2005), who found that age of analyzed individuals and feeding strategy were the most important for Hg accumulation in muscle tissue. It is widely known that fish size is correlated with age. In this study, three specimens weighed more than 5000 grams, of which two were silver carp (Hypophthalmychthys molitrix) with a mercury concentration of 0.446 and 0.436 g/g dry wt. and one was common carp (Cyprinus carpio) with a mercury concentration of 0.342 g/g dry wt. (Table 1, Fig. 3). Those findings support the thesis that it is not necessarily the case that predatory fish are the best indicator species, as previously proposed by Rincon-Leon et al. (1993). They studied the importance of eating habits in the estimation of environmental contamination through indicator species and deduced that omnivorous species such as Cyprinus carpio, as a biological indicator of contamination, allows for estimation with a greater confidence level than the piscivorous species Anguilla anguilla. Similarly }arski et al. (1995) and Svobodova et al. (1999) concluded that benthophagous species feeding close to the bottom are susceptible indicators of environmental contamination depending to the degree of sediment ingestion and direct exposure to contaminated particles. A significant increase in lead concentration was also observed with increasing fish total length (R=0.0003 and p=0.035), while for other metals (Cd and As), no significant relationship with size was determined (p>0.05). The three highest lead concentrations in muscle tissue were recorded in ide (Leuciscus idus); 0.1 and 0.12 g/g dry wt. (Table 3, Fig. 4). Neither of these measured values approaches the maximum permitted level (ML) according to the EU Regulation (1881/2006) and therefore its trends in different fish species were not considered. The data presented here differ from the results obtained by Jari et al. (2011), who measured slightly increased cadmium levels in the muscle of starlet (Acipenser ruthenus) from Danube River in Serbia, while they did not analyse mercury concentrations. In fact, only mercury values were close to or exceeded the ML of 0.5 mg Hg kg-1, indicating a hazard for consumers of fish caught in the Croatian part of the Danube River. In 2003, the Joint FAO/WHO Expert Committee on Food Additives adopted a Provisional Weekly Intake (PTWI) level of 1.6 g/kg weight/week (Smith and Sahyoun 2005), and therefore it would be useful to create an exposure assessment based on the quantity of fish caught either Offical Gazette in commercial fisheries or by angling. Freshwater fish belong to the medium risk group in terms of mean the mercury concentration (ppm) among commercial fish and shellfish (Hansen & Dacher 1997) in Various organ systems can be exposed to low doses of mercury causing damage to the nervous, motoric, renal, and cardiovascular or immune system (Zahir et al. 2005).comparison to some marine fish species like tuna, mackerel or swordfish. Conclusions Among the four metals analysed in the muscle tissue of sampled fish, only mercury exceeded the maximum level permitted according to national and EU regulations in asp, bream and grass carp. Differences between the mean concentrations of measured values in the four groups according to feeding habits were significant. The highest concentration of mercury in an individual specimen was detected in the muscle tissue of asp (Aspius aspius) belonging to the piscivorous fish and a specimen of grass carp (Ctenopharyngodon idella). There was a significant increase in mercury concentration with increasing total length and weight of fish. Due to the increased values of mercury in muscle tissue, it would be useful to establish an exposure assessment based on the quantity of caught and consumed fish either through commercial fisheries or angling. Acknowledgements This study was carried out as a part of the project Management of freshwater fisheries on bordering rivers Pilot study with a holistic regional approach supported by the Croatian Ministry of Agriculture, Forestry and Water Management with the Norwegian partner, Akvaplan-niva AS. 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Low dose mercury toxicity and human health. Environ. Toxicol. Pharm. 20(2), 351 360. }arski, T.P., Debski, B., Beseda, I., Valka, J. (1995). Mercury pollution of bream (Abramis brama L.) caught in the middle course of the Vistula River in 1990 and 1993. Ekol. Bratislava. 14(3), 317-321.     PAGE  PAGE 12  $&*,02>@BFHTVZ\^bdfԿh(0JmHnHuh( h(0Jjh(0JUh>/jh>/Uhy3h.KmH sH hy3hmH sH BDF^`bdf &`#$gdh]hgd90&P1h:p/ =!"#$% DyK zrncic@irb.hryK Bmailto:zrncic@irb.hryX;H,]ą'c @@@ NormalCJ_HaJmHsHtHZ@"Z  Heading 2dd@&[$\$5CJ$\aJ$mH sH tH DA@D Default Paragraph FontRi@R  Table Normal4 l4a (k@(No List HZ@H  Plain TextCJOJ QJ ^J aJtH .X@. Emphasis6]*W@* Strong5\4U@!4  Hyperlink >*phwwwLO2L  enumerationdd[$\$ mH sH tH 0OA0  contribution4 @R4 Footer  !.)@a.  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