Assessment of multiple anthropogenic contaminants and their potential genotoxicity in the aquatic environment of Plitvice Lakes National Park, Croatia.

In this study, the influence of anthropogenic pollution on the aquatic environment of Plitvice Lakes National Park (PLNP) was investigated during 2011-2012 using a combination of chemical and cytogenetic analyses. Four groups of major contaminants [(volatile organic compounds: benzene, toluene, ethylbenzene, and xylenes (BTEX); persistent organochlorine pollutants: organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs); major and trace elements; anthropogenic radionuclides (90Sr, 134Cs, and 137Cs)] were determined in three aquatic compartments (water, sediment, fish). Mass fractions of inorganic constituents in different compartments reflected the geological background of the area, indicating their origin from predominantly natural sources. Levels of volatile and persistent organic compounds in water and fish, respectively, were very low, at levels typical for remote pristine areas. Analysis of anthropogenic radionuclides in water and sediment revealed elevated activity concentrations of 137Cs in water, and measurable 134Cs in the upper sediment layers from April 2011, possibly as a consequence of the Fukushima nuclear accident in March 2011. The potential genotoxicity of river and lake water and lake sediment was assessed under laboratory conditions using the alkaline comet assay on human peripheral blood lymphocytes, and measured levels of primary DNA damage were within acceptable boundaries. The results showed that despite the protected status of the park, anthropogenic impact exists in both its terrestrial and aquatic components. Although contaminant levels were low, further monitoring is recommended to make sure that they will not rise and cause potentially hazardous anthropogenic impacts.

The impact of multiple anthropogenic contaminants on the terrestrial environment of the Plitvice Lakes National Park, Croatia.

The anthropogenic impact on the terrestrial environment of the Plitvice Lakes National Park (PLNP) was investigated through the analysis of three groups of major contaminants (persistent organochlorine pollutants including 15 organochlorine pesticides (OCPs) and 17 polychlorinated biphenyls (PCBs), trace elements/heavy metals (6 major and 23 trace constituents), and anthropogenic radionuclides ((90)Sr, (134)Cs, and (137)Cs)) in three terrestrial compartments (soil, air, and bioindicators of air contamination) during 2011-2013. The correlation coefficients of element mass fractions with soil properties indicated that total Fe and Al minerals, soil organic matter (OM), and organic carbon (OC) content affected the mass fractions of most trace elements in the topsoils. The annual and spatial distributions of heavy metals in total deposited matter (TDM) indicated that the metals came from natural sources and long-range transfer of particulate matter. The PCB and OCP levels found in soil and conifer needles corresponded to global environmental pollution levels by persistent organic pollutants and represented the lower end of the mass fraction ranges reported in the relevant literature. Analyses of anthropogenic radionuclides in bioindicators (conifer needles, lichens, and mosses) showed low but measurable activity concentrations of (134)Cs (for the first time after the Chernobyl accident), which indicated origin from the March 2011 Fukushima Nuclear Power Plant accident. Our overall results indicated that human activity inside or near the PLNP had no significant impact either on contaminant spread by air or on their content in topsoils.

Interrelation between environmental conditions, acanthocephalan infection and metal(loid) accumulation in fish intestine: an in-depth study.

Metal(loid) bioaccumulation in acanthocephalans (Dentitruncus truttae) and intestines of fish (Salmo trutta) from the Krka River, influenced by industrial and municipal wastewaters, was investigated in relation to exposure to metal(loid)s from fish gut content (GC), water, and sediment to estimate potentially available metal (loid)s responsible for toxic effects and cellular disturbances in biota. Sampling was performed in two seasons (spring and autumn) at the reference site (river source, KRS), downstream of the wastewater outlets (Town of Knin, KRK), and in the national park (KNP). Metal(loid) concentrations were measured by ICP-MS. The highest accumulation of As, Ba, Ca, Cu, Fe, Pb, Se and Zn was observed mainly in organisms from KRK, of Cd, Cs, Rb and Tl at KRS, and of Hg, Mn, Mo, Sr and V at KNP. Acanthocephalans showed significantly higher bioaccumulation than fish intestine, especially of toxic metals (Pb, Cd and Tl). Metal(loid) bioaccumulation in organisms partially coincided to exposure from water, sediments and food, while in GC almost all elements were elevated at KNP, reflecting the metal(loid) exposure from sediments. Seasonal differences in organisms and GC indicated higher metal (loid) accumulation in spring, which follows enhanced fish feeding rates. Higher number of acanthocephalans in the intestine influenced biodilution process and lower concentrations of metal(loid)s in fish, indicating positive effects of parasites to their host, as supported by high values of bioconcentration factors. Fish intestine and acanthocephalan D. truttae were confirmed as sensitive indicators of available metal fraction in conditions of generally low environmental exposure in karst ecosystem. Since metal(loid) accumulation depended on ecological, chemical and biological conditions, but also on the dietary habits, physiology of organisms and parasite infection, continuous monitoring is recommended to distinguish between the effects of these factors and environmental exposure when assessing dietary associated metal(loid) exposure in aquatic organisms.

Metal(loid)s and persistent organic pollutants in yellow European eel from the Rasa River, Croatia.

The anthropogenic impact on the aquatic environment of the Rasa River (Croatia) was investigated through the analysis of seven polybrominated diphenyl ethers (PBDEs), seven polychlorinated biphenyls (PCBs), three DDT isomers, and 22 major and trace elements using yellow European eel (Anguilla anguilla L.) as a biological indicator of contamination. The obtained data indicated generally low contamination status in the surrounding area. Levels of all organic contaminants in muscle significantly increased with lipid content, length, weight and body condition. In both muscle and liver, most metal(loid)s decreased or remained unchanged with increasing size, while at downstream location only several elements (Cd, Cu, Fe, Na, Se, U, V, Zn) accumulated in the liver with fish growth. Spatial analysis revealed higher pressure of Ag, Cd, Cr, Mo, Tl, U, and V at the downstream location, revealing the potentially limited impact of historical coal mining industry on the lower reaches of the Rasa River.

Effects of Maternal Cigarette Smoking on Trace Element Levels and Steroidogenesis in the Maternal-Placental-Fetal Unit.

This study evaluates the interaction of toxic elements cadmium (Cd) and lead (Pb) due to exposure from cigarette smoking, essential elements, and steroidogenesis in the maternal-placental-fetal unit. In a cohort of 155 healthy, postpartum women with vaginal term deliveries in clinical hospitals in Zagreb, Croatia, samples of maternal blood/serum and urine, placental tissue, and umbilical cord blood/serum were collected at childbirth. The biomarkers determined were concentrations of Cd, Pb, iron (Fe), zinc (Zn), copper (Cu), and selenium (Se), and steroid hormones progesterone and estradiol in maternal and umbilical cord blood and the placenta. Three study groups were designated based on self-reported data on cigarette smoking habits and confirmed by urine cotinine levels: never smokers (n = 71), former smokers (n = 48), and active smokers (n = 36). Metal(loid)s, steroid hormones, urine cotinine, and creatinine levels were analyzed by ICP-MS, ELISA, GC-MS, and spectrophotometry. Cigarette smoking during pregnancy was associated with increased Cd levels in maternal, placental, and fetal compartments, Pb in the placenta, and with decreased Fe in the placenta. In active smokers, decreased progesterone and estradiol concentrations in cord blood serum were found, while sex steroid hormones did not change in either maternal serum or placenta. This study provides further evidence regarding toxic and essential metal(loid) interactions during prenatal life, and new data on sex steroid disruption in cord serum related to cigarette smoking. The results indicate that umbilical cord sex steroid levels may be a putative early marker of developmental origins of the future burden of disease related to harmful prenatal exposure to cigarette smoke.

Potentially Toxic Elements in Water, Sediments and Fish from the Karstic River (Rasa River, Croatia) Located in the Former Coal-Mining Area.

The assessment of the environmental quality of a sensitive karst aquatic system under the centuries-long anthropogenic influence of the coal mining industry is important for both improving the quality of water resources and protecting aquatic wildlife and human health. In this study, we investigated the anthropogenic impact on the aquatic environment of the upper and middle course of the Rasa River through the analysis of a suite of metal(loid)s in three aquatic compartments (water, sediment, fish) using inductively coupled plasma mass spectrometry (ICP-MS). Concentrations of inorganic constituents in water were low, while the chemical composition of stream sediments mainly reflected the geological background of the area, indicating the origin of metal(loid)s from predominantly natural sources. Although comparison with PEC-Q values indicated that existing sediment quality conditions could pose a threat to benthic organisms with regard to Cr and Ni, the constant vertical profiles of these elements suggested their natural origin from the weathering of flysch. Element levels in the muscle of targeted fish species were in accordance with the values typical for low-contaminated freshwater systems, while levels of Cd, Pb and Hg were mostly below the European regulatory limits for toxic elements in foods, indicating that the low concentrations of most contaminants in muscles of fish from the Rasa River do not present a risk to humans or other consumers. The obtained data indicated a generally low contamination status of the western part of the Rasa River basin with regard to the analyzed inorganic elements.

The effects of maternal cigarette smoking on cadmium and lead levels, miRNA expression and biochemical parameters across the feto-placental unit.

Several miRNAs have been previously identified to be associated with cigarette smoke and/or the toxic metals cadmium (Cd) and lead (Pb). The aim of this study was to investigate the associations of maternal cigarette smoking with cadmium (Cd) and lead (Pb) levels, candidate miRNA expression and biochemical parameters across the feto-placental unit. miRNAs were isolated according to protocols provided by manufacturer from 72 healthy postpartum women using Qiagens' kits based on phenol/guanidine samples lysis and silica-membrane purification of total RNA. Candidate miRNAs (miR-1537, miR-190b, miR-16, miR-21, and miR-146a) were quantified by real-time PCR. Biochemical parameters were analyzed in plasma samples by standardized and harmonized enzymatic methods using appropriate calibrators, while CRP was determined by immunoturbidimetric method. Concentration of Cd and Pb in whole blood and placenta samples were measured by inductively coupled plasma mass spectroscopy. Cd levels in smokers were higher in all of the analyzed compartments of the feto-placental unit, Pb in maternal blood and placenta than non-smokers. Smokers also had a higher expression of miR-16 in maternal and miR-146a in cord plasma, and lower expression of miR-21 in the placenta in comparison to non-smokers. Urate concentrations in the maternal plasma of smokers were lower than this value in non-smokers. The study has demonstrated that maternal smoking was associated with toxic metals (Cd and Pb) levels, urate concentration and alteration of miRNA expression. Given that the effects of maternal smoking on miRNA expression are inadequate, all compartments of the feto-placental unit should be analyzed to obtain a complete picture. This paper is the first to report on the results of expression of cellular and circulating miRNAs simultaneously in maternal and fetal compartments and in the placenta.

Distribution of polychlorinated biphenyls and organochlorine pesticides in wild mussels from two different sites in central Croatian Adriatic coast.

Levels of 24 organochlorine compounds were investigated in wild mussels collected at two locations (Krka estuary and Kastela Bay) on the Croatian Adriatic coast in 2003, 2005, 2006, 2007, and 2008. PCB and OCP ranges found at the two locations overlapped and followed similar profiles despite the differences between the two locations indicating that they share a common pollutants source. Among organochlorine pesticides, the dominant compound was DDT. Among indicator PCBs, the dominant compound was PCB-153, while PCB-118 was dominant among the remaining 11 congeners. Generally, the sum of six indicator PCBs was constantly greater than the sum of 11 congeners at both locations. α-HCH/γ-HCH and DDE/DDT ratios were below 1, indicating recent input of γ-HCH and DDT into the marine environment. In the investigated period, almost all organochlorine compounds reached the highest values in 2006. The levels of PCBs and OCPs in this study were considerably below the Croatian maximum permissible levels, confirming that they do not pose any threat for human health.

Mercury and selenium levels in archive samples of wild Atlantic bluefin tuna from the Mediterranean Sea.

This study examined total mercury (THg) and selenium (Se) levels in archive samples (white and red muscles, liver, gills) of the wild Atlantic bluefin tuna (ABFT) (Thunnus thynnus) (n = 18) captured in the central Adriatic Sea. The influence of fish size, age, and tissue type on element distribution was examined. There were significant differences in THg and Se levels, and Se:THg molar ratios among tissues. THg levels were highest in liver and lowest in gills (liver > red muscle > white muscle > gills), while Se levels were also highest in liver but lowest in white muscle (liver > red muscle > gills > white muscle). Se:THg molar ratios were highest in gills (22-82), intermediate in liver (11-29) and red muscle (7-36), and lowest in white muscle (1.7-7.6). Concentrations of THg in all tissues and Se in liver and caudal muscle were positively correlated with tuna age and size, while the Se:THg molar ratio in gills and all white muscles was negatively correlated with tuna age and size, indicating that the protective role of Se against THg is reduced in older specimens. The selenium health benefit values (HBVSe) were above zero in all tissues, indicating a small excess of Se after Hg sequestration. However, since the obtained HBVSe for edible tissues were near zero (0.01-0.04), and more than 70% of white muscle samples and all red muscle samples exceeded the EU regulatory limit for THg in fish muscle, it would be advisable to limit their intake in adults to one meal per month.

Evaluation of lead exposure in battery-manufacturing workers with focus on different biomarkers.

The influence of exposure to lead on the frequency of micronuclei (MN), nuclear buds and nucleoplasmatic bridges was investigated in peripheral blood lymphocytes in 15 male battery-manufacturing workers and 15 controls matched for age and smoking habits. In addition to MN test, blood lead (B-Pb) and cadmium (B-Cd), delta aminolevulinic acid dehydratase (ALAD) activity, erythrocyte protoporphyrin (EP), serum vitamin B(12) (S-Vit B(12)) and folate (S-folate) were determined in all subjects. Lead-exposed subjects had significantly higher MN frequency and B-Pb concentrations than controls. In control smokers we found a significant negative correlation between B-Pb concentration and frequency of nucleoplasmatic bridges, and nuclear division index. In control non-smokers a significant positive correlation was observed only between age and nuclear buds frequency, and between S-folate and B-Pb level. In lead exposed smokers, significant positive correlations between MN frequency and S-Vit B(12), S-folate, and nuclear buds frequency were found. A positive correlation in exposed smokers was also found between nuclear buds frequency and S-Vit B(12) concentration. A negative correlation was found between ALAD and EP, and B-Pb in exposed smokers. Exposed non-smokers showed significant negative correlation between MN frequency and B-Cd, and ALAD and EP. The results indicate a genotoxicity of lead, pointing to a micronucleus assay as a relevant test for assessing genotoxic effects resulting from occupational exposure. The other indicators did not necessarily follow the results of THE MN test. Influence of smoking should be further investigated.

Legacy persistent organic pollutants (POPs) in archive samples of wild Bluefin tuna from the Mediterranean Sea.

Polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) were quantified in archive samples of dorsal white muscle of the wild Bluefin tuna (BFT) (n = 9) collected in the central Adriatic. PCBs were the predominant organochlorine (OC) compounds (60.8-69.4% of the total OC load) found. The contribution of HCB and ΣHCHs was significantly lower, ranging from

Mercury and selenium in fish from the eastern part of the Adriatic Sea: A risk-benefit assessment in vulnerable population groups.

The study examines the levels of total mercury (Hg) and selenium (Se) in the muscle tissue of 12 commercially important fish species (n = 717) from 48 locations in the eastern Adriatic Sea, as a function of fish species, size and habitat. Risks and benefits of fish consumption regarding Hg and Se in children (7 years) and women of reproductive age (30 years) were also evaluated. All samples but one were compliant with the European regulatory limit for Hg in fish muscle. Concentrations (µg g-1 ww) of Hg (range: 0.014-1.37) and Se (range: 0.184-1.12) and Se:Hg molar ratios in fish were associated with habitat type. Demersal species had the highest Hg and the lowest Se concentrations, while pelagic species had the lowest Hg concentrations, highest Se concentrations and Se:Hg molar ratios. All species had Se:Hg molar ratios above 1 (range: 1.4-66.9) and a selenium health benefit value (HBVSe) above zero (range: 3.3-9.3), indicating Se excess after Hg sequestration. The intake of two meals per week of small pelagic-neritic and bento-pelagic fish is estimated to be a valuable source of essential Se, and does not pose a risk from toxic Hg exposure in children and women during the vulnerable reproductive period. Due to the low Hg and favorable Se:Hg molar ratio and HBVSe index, small pelagic species offer a good risk to benefit ratio of fish consumption and could be considered an optimal choice in these sensitive subpopulations.

Biomonitoring findings for occupational lead exposure in battery and ceramic tile workers using biochemical markers, alkaline comet assay, and micronucleus test coupled with fluorescence in situ hybridisation.

Manufacture of lead-containing products has long been associated with various health risks. To get an insight into the related genotoxic risks, we conducted a biomonitoring study in 50 exposed workers and 48 matched controls using a battery of endpoints that sensitively detect the extent of genome instability in peripheral blood lymphocytes. The levels of primary DNA damage were estimated with the alkaline comet assay, while cytogenetic abnormalities were determined with the cytokinesis-block micronucleus (CBMN) cytome assay. Additionally, CBMN slides of 20 exposed and 16 control participants were subjected to fluorescence in situ hybridisation (FISH), coupled with pancentromeric probes to establish the incidence of centromere-positive micronuclei, nuclear buds, and nucleoplasmic bridges. Blood lead levels (B-Pb) were measured with atomic absorption spectrometry. To further characterise cumulative effects of occupational exposure, we measured erythrocyte protoporphyrin (EP) concentrations and delta-aminolevulinic acid dehydratase (ALAD) activity in blood. We also assessed the influence of serum folate (S-folate) and vitamin B12 (S-B12) on genome stability. Compared to controls, occupationally exposed workers demonstrated significantly higher B-Pb (298.36±162.07 vs 41.58±23.02), MN frequency (18.71±11.06 vs 8.98±7.50), centromere positive MN (C+ MN) (8.15±1.8 vs 3.69±0.47), and centromere negative MN (C- MN) (14.55±1.80 vs 4.56±0.89). Exposed women had significantly higher comet tail intensity (TI) and length (TL) than control women. Furthermore, workers showed a positive correlation between age and nuclear buds and MN, between MN and years of exposure, and between S-B12 levels and TI and ALAD activity, while a negative correlation was found between TI and B-Pb. These findings suggest that occupational settings in the manufacture of lead-containing products pose significant genotoxic risks, which calls for developing more effective work safety programmes, including periodical monitoring of B-Pb and genetic endpoints.

Levels of organochlorine compounds in the Mediterranean blue mussel from the Adriatic Sea.

The distribution of polychlorinated biphenyls (PCBs) and organochlorine pesticides (OCPs) was investigated in Mediterranean blue mussel collected at four locations in Mali Ston Bay few times a year in 2005­2007. OCPs were found in all samples and levels ranged between 0.07 and 7.58 ng g−1 dry wt. Levels of PCBs ranged between 0 (below detection limit) and 21.55 ng g−1 dry wt. For most analyzed compounds there were no significant level changes between the 3 years. Exceptions are decreased levels of ß-HCH, DDD, and PCB-138 and increased levels of γ-HCH and DDT in 2007. However, mussels from this area are applicable for human diet.

Impact of fish farming on the distribution of phosphorus in sediments in the middle Adriatic area.

During the last decade, intensive fish farming developed along the central Croatian coast, creating a need to study and evaluate its potential influence on unaffected sites. We considered phosphorus as an indicator of the influence of fish farming and investigated the distribution of phosphorus forms in sediment from several fish farms and marine areas of different trophic status in the middle Adriatic. Analyses of samples were performed with modified SEDEX techniques. Our results indicated that authigenic apatite phosphorus showed no significant differences among the investigated stations, while organic phosphorus concentrations reflected the trophic status of the station area. Below-cage sediment was characterized by enhanced fish debris phosphorus and low detrital apatite phosphorus concentrations, while sediment from an anthropogenically influenced bay showed the highest values of iron bound phosphorus species. Among the different P fractions, fish debris phosphorus proved to be the most sensitive indicator of the influence of fish farming on marine sediment.

Biomonitoring of mercury in polluted coastal area using transplanted mussels.

The Kastela Bay is heavily polluted with inorganic mercury originated from direct discharges from the chlor-alkali plant, which operated in the period from 1950 to 1990. Even though the plant was closed 15 years ago, elevated levels of total mercury are still evident in surface sediments of the bay. In order to assess the availability of remobilized mercury to marine organisms, cultured mussels (M. galloprovincialis) were transplanted from pristine area to Kastela Bay, in the period from September 2000 to March 2001. Mussel samples were collected for the analysis of THg and MeHg in whole soft tissue, gills and digestive gland. Surface sediments and suspended matter were collected for the analysis of THg. Digestive gland was the target organ for the accumulation of THg, while concentrations of MeHg were similar in all analyzed tissues. The percentage of MeHg in mussel tissues (4-27%) was characteristic for the areas contaminated with inorganic mercury. A significant negative correlation was observed between the THg concentration in the tissues and the percentage of MeHg. Concentrations of THg in mussel tissues, which were decreasing from the source of contamination in an anticlockwise direction towards the exit of the bay, were significantly positively correlated to THg content in sediment and suspended particles. Spatial distribution of mercury species (THg and MeHg) in different environmental compartments was in accordance with the prevailing circulation in the bay. Data obtained through 6 months of biomonitoring experiment indicated that digestive gland was more sensitive indicator of THg concentrations in the environment than the whole organism or gills. As for MeHg, all tissues were equally suitable as biomonitors of MeHg concentrations in the environment.

Chlorinated compounds in the muscle tissue of fish from the Croatian Adriatic: preliminary data on contamination and the associated health risks.

Levels of 17 polychlorinated biphenyl congeners (PCBs) and seven organochlorine pesticides (OCPs) were measured in the muscle tissue of 18 commercially important fish species from the eastern Adriatic Sea. PCBs [

Organochlorine pesticides and polychlorinated biphenyl congeners in wild terrestrial mammals from Croatia: Interspecies comparison of residue levels and compositions.

In this pilot study, we investigated levels of organochlorine pesticides (OCPs) and polychlorinated biphenyls (PCBs) in the adipose tissues of two free-ranging terrestrial carnivores from Croatia sampled in 2010 and 2011: the brown bear (Ursus arctos; N=32) and the grey wolf (Canis lupus; N=29). Concentrations of ∑OCPs and ΣPCBs ranged from 0.45 to 4.09 ng g(-1) lipid mass (lm) and from 0.93 to 8.52 ng g(-1) lm in brown bear, and from 1.18 to 5.67 ng g(-1) lm and 2.68 to 48.9 ng g(-1) lm in grey wolf adipose tissues, respectively. PCBs were dominant accounting for over 72% of total analyzed persistent organic pollutants (POPs). The sum of six indicator non-dioxin-like PCBs (Σ6IndNDL PCBs) made up 60-93% and 58-85% of the total congener concentrations in brown bears and wolves, respectively. Although the levels of the measured parameters were significantly higher in grey wolves than in bears, the contaminant profiles of the two species were similar, with γ-HCH, HCB, ß-HCH and DDE as major OCP contaminants, and PCB-153>PCB-180≈PCB-170>PCB-138 as the dominant congeners. The sum of hexachlorocyclohexanes (ΣHCHs) and 8 toxicologically relevant dioxin-like PCBs (Σ8ToxDL PCBs) was higher in the males than in the females of the brown bear. Concentrations of ΣDDTs, HCB, ΣOCP, ΣPCBs, Σ6IndNDL PCBs, and toxicologically relevant non-dioxin-like PCBs (ΣToxNDL PCBs) were significantly positively correlated with lipid content in the grey wolf. Concentrations of OCPs and PCBs in brown bears and wolves from Croatia were low and normal for large terrestrial mammals.

Distribution of persistent organic pollutants (POPs) in cultured mussels from the Croatian coast of the Adriatic Sea.

In this study we investigated the distribution of 7 organochlorine pesticides (OCPs) and 17 polychlorinated biphenyl (PCBs) congeners in the edible tissue of the blue mussels (Mytilusgalloprovincialis) collected at 15 shellfish breeding farms and 1 harvesting area along the Croatian Adriatic coast. All analyzed OCPs were found in all samples (0.011-1.47 ng g(-)(1) w.wt.). Concentrations of PCB congeners in positive samples ranged between 0.007 and 7.66 ng g(-)(1) w.wt. The most abundant compounds were γ-HCH, PCB-138 and PCB-153. Overall levels of PCBs and OCPs were in the lower end of the concentration ranges reported in literature. Significant differences of all contaminants were recorded between seasons, with higher values in the warmer part of the year. Results of the evaluation of the risks to human health associated with consumption of the mussels containing organic contaminants suggest that the levels of these compounds in mussels do not pose any threat for consumers of cultivated mussels.

Cadmium and lead in grey wolf liver samples: optimisation of a microwave-assisted digestion method.

A microwave-assisted digestion method for the determination of cadmium (Cd) and lead (Pb) by inductively coupled plasma mass spectrometry (ICP-MS) was optimised on certified reference material (CRM) (bovine liver, BCR-185R) and wolf liver samples. Different factors influencing digestion efficiency (temperature, time, composition of the digestion mixture, sample mass) were tested. Validation included linearity (up to 200 µg L(-1) for Cd and Pb), detection (0.003 µg L(-1) for Cd and 0.035 µg L(-1) for Pb), and quantification (0.008 µg L(-1) for Cd and 0.081 µg L(-1) for Pb) limits. Good agreement between measured and certified values was achieved in all conditions, with recoveries ranging from 94 % to 111 % for Cd and from 95 % to 105 % for Pb. The precision of the method, expressed as relative standard deviation, was up to 3 % for Cd and 8 % for Pb. The best digestion parameters (260 °C, 30 min, 1 mL HNO3+4 mL H2O, 0.1 g of CRM) based on accuracy and precision were applied on two wolf liver samples to evaluate the need for the predigestion step (freeze-drying) and appropriate mass of the sample. Freeze-drying improved precision and minimising the tissue mass to 0.1 g reduced the matrix effect. Using these optimised digestion conditions, we determined Cd and Pb in 40 wolf livers collected in Croatia, and their medians (0.055 µg g(-1) and 0.107 µg g(-1), respectively) were in the range of previously reported data for the grey wolf.