Pesticides and Polychlorinated Biphenyls in Milk and Dairy Products in Croatia: A Health Risk Assessment.

The aim of this study was to evaluate contamination levels and the frequency of detection of organochlorine (OCPs) and organophosphate pesticides (OPPs), pyrethroids, carbamates and polychlorinated biphenyls (seven PCB congeners) in a total of 534 samples of cow's, sheep's and goat's milk and dairy products from Croatia. Concentrations above the limit of quantification (LOQ) were measured for fourteen OCPs, nine OPPs, six pyrethroids, one carbamate and PCBs with a total of 172 results, and no concentrations exceeded the maximal residue levels defined by the European Union. The mean concentrations of pesticides and the sum of seven PCBs were determined in the ranges 0.92-17.4 µg/kg and 1.38-2.74 µg/kg. Pesticides were quantified in 27% of samples, and seven PCBs were quantified in 5.23% of samples. Among the three pesticide groups, the highest numbers of quantified results were found for OCPs (12.1-20.8%). The highest frequencies of detection were found for the sum of 4,4'-dichlorodiphenyltrichloroethane and its isomers (DDTs), hexachlorobenzene and seven PCBs. The sum of seven PCBs was quantified within the range of 3.3-6.67% of samples per milk type and dairy products. Among the OPPs, the highest frequency of detection was found for chlorpyrifos in cow's milk. Based on the estimated daily intakes, chronic risk characterisation showed no risk for adults or ten-year-old children for the consumption of cow's milk and dairy products.

Distribution of Pesticides and Polychlorinated Biphenyls in Food of Animal Origin in Croatia.

Pesticides and polychlorinated biphenyls (PCBs) are persistent environmental pollutants. When entering the food chain, they can represent a public health problem due to their negative effects on health. In this study, concentrations of organochlorine pesticides (OCPs), organophosphate pesticides (OPPs), pyrethroids, carbamates, and PCBs-a total 73 compounds-were determined in a total of 2268 samples of fat tissues (beef, pork, sheep, goat, poultry, game, horse, rabbit) and processed fat, meat, and processed meat products collected in Croatia during an 8-year period. In fatty tissues, 787 results exceeded the limits of quantification (LOQ): 16 OCPs, eight OPPs, six pyrethroids, one carbamate, and seven PCBs. The most positive results in fat samples were found for OCPs, with a frequency of quantification in the range of 57.5-87.5%. Hexachlorobenzene (HCB) and dichlorodiphenyldichloroethylene (DDE) were quantified in the highest percentages, in the ranges of 5.5-66.7% and 5.4-55.8%. Concentrations above the MRL values were determined for chlorpyrifos in pork fat and for resmethrin in six fat samples and one pâté. In 984 samples of meat and meat products, only 62 results exceeded the LOQ values. The highest frequency of quantification was determined for OCPs (25 samples), of which 40% were DDT isomers (60% DDE). Frequency quantifications of PCBs in fat samples were between 7.23 and 36.7%. An evaluation of the health risk assessment showed that the consumption of fat, meat, and meat products does not pose a threat to consumer health, since all EDI values were well below the respective toxicological reference values.

Body burdens and distribution of mercury and selenium in bottlenose, striped and Risso's dolphins along the Adriatic coast: A 20-year retrospective.

Top marine predators present high mercury concentrations in their tissues due to biomagnification in the marine food chain. This study reports mercury (Hg) and selenium (Se) status, and the Hg:Se molar ratio assessment in bottlenose (Tursiops truncatus), striped (Stenella coeruleoalba) and Risso's dolphins (Grampus griseus). Total Hg and Se concentrations were determined in muscle, liver, kidney, lung, spleen, adipose tissue and skin collected from 186 specimens stranded in the Croatian part of Adriatic Sea from 1995 to 2014. Total Hg concentrations in tissue samples ranged from 0.001 in the spleen to 2238 mg/kg wet weight in liver. Se concentrations in dolphin samples ranged from 0.010 to 2916 mg/kg ww. Minimum Se concentration was found in muscle and maximum Se concentration were found in liver of bottlenose dolphin. Hg and Se levels in Risso's dolphins showed higher concentrations in all tissues in comparison to bottlenose and striped dolphins. Significant and positive correlations were observed between age and Hg concentrations (P < 0.05). In 66.6 % of Risso's, 15.3 % of bottlenose dolphins and one stranded striped dolphin in this study, the hepatic concentration of Hg exceeded the higher toxic thresholds (400 mg/kg w.w.) previously defined as evidence of liver damage in marine mammals. The Hg:Se molar ratio in the liver of Risso's dolphin was 0.670. The liver of adult bottlenose dolphins showed expected values (0.870), while the liver of young dolphins had a high ratio (0.750), non-specific for the age group. The Hg:Se molar ratio in the liver of striped dolphins was 0.390, which is lower than the literature values.

Seasonal Occurrence of Aflatoxin M1 in Raw Milk during a Five-Year Period in Croatia: Dietary Exposure and Risk Assessment.

This study's objective was to estimate the seasonal occurrence of aflatoxin M1 (AFM1) in cow's milk between winter 2016 and winter 2022 and to assess dietary exposure and risk assessment for the adult Croatian population. In total, 5817 cow milk samples were screened for AFM1 concentrations using the enzyme immunoassay assay (ELISA). For confirmation purposes of AFM1 concentration above the European Union maximum permitted level (MRL), ultra high-performance liquid chromatography with tandem mass spectrometry was performed. In 94.7% of milk samples, AFM1 levels were below the detection limit (LOD) of the ELISA test. For 3.47% of samples, the AFM1 was between the LOD and MRL values. Only 1.87% of all samples exceeded the MRL. The mean value of elevated AFM1 in different seasons ranged between 59.2 ng/kg (autumn 2017) and 387.8 ng/kg (autumn 2021). The highest incidences of positive AFM1 were determined in autumn and winter and the maximum (6.4%) was in winter 2019/2020. The largest percentage of positive samples (69.7%) was found in central Croatia. The estimated daily intakes for positive samples ranged between 0.17 and 2.82 ng/kg body weight/day. Risk assessment indicated a high level of concern during autumn and winter, especially for consumers of large amounts of milk.

The Content of Cobalt, Silver and Vanadium in Raw Cow's Milk in Croatia and Risk Assessment for Consumers.

Concentrations of selected trace elements Ag, Co and V in raw milk sampled from four geographical regions in Croatia were measured. Silver, Co and V were detected above the limit of detection within the range of 9.52%-30.8%, 1.6%-12.1% and 12.4%-30.8%. Silver concentrations were not detected in milk samples from the Croatian Littoral and Mountainous Croatia (CL-MC) region. Similar Ag content was found in Southern, Eastern and Central Croatia. The lowest mean of Co and V of 33.2 and 83.8 µg kg-1 were found in the CL-MC region while the highest of 49.8 and 136.9 µg kg-1 was found in Central Croatia. There were no statistically significant differences in Ag, Co and V contents between the four regions. The estimated daily dietary intakes (EDI) of total mean and total 95th percentile values of Ag, Co and V showed lower values in comparison with available EFSA health-based limits.

Difference in pesticides, trace metal(loid)s and drug residues between certified organic and conventional honeys from Croatia.

Quality and safety of food, including honey, is one of the leading priorities regarding residues of anthropogenic chemicals with proven adverse health effects. In total, 61 honey samples of known botanical origin were collected in period 2018-2019 from Croatian registered organic and conventional beekeepers (N = 16 organic and N = 45 conventional honey samples). Eleven trace metal(loid)s (Al, As, Cd, Co, Cr, Cu, Fe, Mn, Ni, Pb and Zn; quantitated by ICP-MS), 24 antibiotics (LC-MS/MS, ELISA, microbiological inhibition test), six indicator PCBs (GC-MS/MS) and 121 pesticides (LC-MS/MS, GC-MS/MS) originating from environment and/or beekeeping practice were measured to assess possible differences in contaminant residues between organic and conventional honeys. All honey samples had contaminant residues below the legal maximum levels and are considered safe for consumers. However, 2/16 organic and 34/45 conventional honeys contained one or two synthetic acaricides (most frequently coumaphos), while other pesticides, antibiotics and PCBs were not quantified. Also, organic honey contained lower levels of coumaphos, amitraz and amitraz metabolite N-(2,4-dimethylphenyl) formamide than conventional honeys, on average. Higher levels of Cr (p = 0.006) were detected in organic compared to conventional chestnut honeys. This study pinpointed beehive disease control treatment as prominent pesticide residue source, which was to some extent reduced in organic honeys. Quantified pesticide and metal(loid) levels were within range or lower than reported in recent literature regarding honey of the same botanical origin.

Evaluation of Element Concentrations in Beef and Pork Meat Cuts Available to the Population in the Croatian Capital.

The aim of this study was to determine the concentrations of essential, trace, and toxic elements in beef and pork meat cuts available at markets and retail chains in the Croatian capital. Significant differences in the concentrations of Al, Cr, Cu, Fe, Mg, Mn, Mo, Ni, Se, Pb, and Zn were found between bovine cuts (p < 0.01, all) and also between pork cuts (p < 0.01, all). A risk assessment using the estimated intakes based on the lowest and highest mean values of Al, Cr, Ni, and Pb in beef and pork showed low contributions to tolerable toxicological limits. However, consumers whose diets consist of large amounts of beef and pork kidneys may be at risk because the estimated intakes for Cd and Se exceeded the toxicological limits. Consumers of large quantities of beef mixed meat may be at risk due to higher values of estimated As intakes compared to health-based guidance values. Estimation based on the provisional maximum tolerable daily intake values for Cu, Fe, and Zn showed that beef and pork cuts can be considered safe for consumption. A comparison with data from other studies shows that the concentrations of the analyzed elements in beef and pork cuts vary considerably.

Assessment of Toxic and Trace Elements in Multifloral Honeys from Two Regions of Continental Croatia.

Element concentrations were measured in multifloral honeys sampled from Central and Eastern Croatia. The mean levels of elements ranged from (µg/kg): Al 323-7228, Cu 103-1033, Cr 14.4-139, Fe 295-2336, Ni 122-523, Pb 9.65-154, Zn 442-2025. In all samples, As and Cd content were below the LOD values. Significant differences in the concentrations of Al, Cr, Cu, Fe, Pb and Zn (p < 0.01) were found in honeys from different locations within regions and within locations of each region. Also, significant differences in total element contents between the two regions were determined for Cr and Cu (p < 0.01). No significant differences were observed in total Al, Fe, Pb and Zn levels between regions. The highest Al, Cr, Cu, Fe and Zn concentrations were measured in Central Croatia, while Ni and Pb in Eastern Croatia. The results confirm the decisive influence of collection location on the composition of toxic and trace elements in honey.

Manganese Concentrations in Tissues and Skin of Three Dolphin Species Stranded in the Croatian Waters of the Adriatic Sea from 1995 to 2013.

Manganese concentrations were determined in muscle, kidney, liver, spleen, lung and fat tissues and skin of three cetacean species, the bottlenose (Tursiops truncatus), striped (Stenella coeruleoalba) and Risso's (Grampus griseus) dolphins, stranded along the Croatian Adriatic coast between 1995 and 2013. Mean ranges determined in tissues were (mg/kg, ww): muscle 0.23-0.27, liver 2.87-4.00, kidney 0.66-1.26, spleen 0.37-0.79, lung 0.18-0.57, skin 0.11-0.97, fat 0.06-0.42. The highest Mn mean levels in tissues were measured in species (mg/kg, ww): T. truncates lung 0.41, skin 0.97, fat 0.42; S. coeruleoalba muscle 0.26; G. griseus kidney 1.26, liver 4.00, spleen 0.64. Significant differences of Mn concentrations in the liver (p = 0.034), spleen (p = 0.037) and skin (p = 0.013) were found among the three dolphin species. Significant differences in Mn levels were found between young and adult T. truncates in kidney (p = 0.042), lung (p = 0.0040) and skin (p = 0.0034).

Essential and toxic element concentrations in monofloral honeys from southern Croatia.

The concentrations of 24 elements in seven honey types (multifloral, heather, common heather, bearberry, sage, mandarin orange-blossom and honeydew) collected in southern Mediterranean regions of Croatia were determined using ICP-MS. Significant differences were found in the concentrations of Ag, As, Ba, Cu, Co, Fe, K, Mg, Mn, Mo, Na, Ni, Se, Sb, U and Th (p<0.05, all) among honeys. The highest element concentrations were determined in honeydew honeys, with the exception of multifloral (Ca, Cr, Mo, Se), common heather (Mg, Na), bearberry (Ba, Fe, Pb) and sage (Ag) honeys. Among the floral honeys, the highest concentrations were found in multifloral honey (Al, As, Be, Ca, Cr, Mn, Mo, Ni, Se, Th and U), common heather (Co, K, Mg, Na, V), sage (Ag, Cd, Cu), and bearberry (Ba, Fe, Pb, Sb, Zn). The results contribute to the evidence supporting the role of botanical origin on the elemental composition of honey.

Copper Levels in Tissues of Dolphins Tursiops truncatus, Stenella coeruleoalba and Grampus griseus from the Croatian Adriatic Coast.

Copper concentrations were determined in muscle, liver, kidney, spleen and lung tissues of three dolphin species. Dolphins of Tursiops truncatus (young and adult), Stenella coeruleoalba and Grampus griseus were stranded along the Croatian coast. Concentrations in tissues of all three dolphin species were highest in the liver (4.92-16.5 µg/g) followed by kidney (2.85-5.29 µg/g). Similar levels were measured in muscle, spleen and lung in range 1.13-3.67 µg/g. Statistics analysis showed significant differences of Cu concentrations for muscle (p = 0.008), kidney (p = 0.04) and liver (p = 0.02) between the three dolphin species. Also, for all three species significant differences between tissue types of the same species were determined (p < 0.001, all). However, there were no significant differences in Cu levels of the same tissues between males and females within same species. Also, significant differences of body length and weight between three dolphin were found (p < 0.001, both).

Validation of a liquid chromatography-tandem mass spectrometry method for the simultaneous determination of sulfonamides, trimethoprim and dapsone in muscle, egg, milk and honey.

A quantitative multi-residue method that includes 13 sulfonamides, trimethoprim and dapsone was developed and validated according to Commission Decision 2002/657/EC for muscle, milk egg and honey samples. For all matrices, the same extraction procedure was used. Samples were extracted with an acetone/dichloromethane mixture and cleaned up on aromatic sulfonic acid (SO3H) SPE cartridges. After elution and concentration steps, analytes were identified and quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Data were acquired according to the multiple reaction-monitoring approach (MRM) and analytes were quantified both by the isotope dilution and the matrix-matched approaches calculating the response factors for the scanned product ions. The developed method shows good linearity, specificity, precision (repeatability and within-laboratory reproducibility), and trueness. Estimated CCß for sulfonamides ranged between 5.6 and 8.2 µg kg(-1) for eggs, between 11.1 and 69.9 µg kg(-1) for milk, between 64.7 and 87.9 µg kg(-1) for muscle, and between 2.7 and 5.3 µg kg(-1) for honey. CCß values for dapsone were 3.1, 0.6, 0.7 and 1.5 µg kg(-1) and for trimethoprim were 3.1, 6.7, 81.7 and 3.0 µg kg(-1) calculated for eggs, milk, muscle and honey, respectively. Recovery for all matrices was in the range from 89.1% and 109.7%. In matrix effect testing, no significant deviations were found between different samples of muscle and milk; however, a matrix effect was observed when testing different types of honey. The validation results demonstrate that the method is suitable for routine veterinary drug analysis and confirmation of suspect samples.

Lead Concentrations in Raw Cow and Goat Milk Collected in Rural Areas of Croatia from 2010 to 2014.

A total of 249 cow and 33 goat milk samples were collected in rural areas of Croatia during the period 2010-2014. Lead concentrations in milk samples were analyzed by graphite furnace-atomic absorption spectroscopy. Mean Pb concentrations in milk ranged from (µg/kg): cow 10.8-12.2; goat 9.33-60.0. The highest Pb level of 131 µg/kg in cow milk was measured during 2014. There were no significant differences in Pb levels between cow and goat milk and also in goat milk among the analysed years. However, significant differences were found in cow milk among years. The highest Pb was determined in 2011 (157 µg/kg in goat milk). The calculated estimated weekly intakes of Pb concentrations for cow and goat milk contribute only 1.37 % and 1.84 % to the provisional tolerable weekly intake. Therefore, the consumption of milk from both species should not pose a consumer health risk.

Balance of glucosinolates content under Cd stress in two Brassica species.

We designed a pot experiment to study the changes of growth parameters, accumulation of Cd in cabbage and kale, during a prolonged exposure period with two harvests (after 45 and 75 days of plant growth). Simultaneously, we monitored changes of total S and GSL contents in different plant organs. Both Brassica species showed tolerance to Cd, since no obvious symptoms of metal toxicity were displayed and no significantly reduced plant biomass were recorded. Moreover, a trend of root biomass enhancement was noticed. In general, the Cd concentration in roots was higher than in other organs. Data indicate that the transport system from roots to shoots was already saturated after the first growing period, while root continued to accumulate Cd during the second growing period. Regardless of differences in the GSL content between cabbage and kale, both Brassica species had a similar response to Cd exposure, indicating that the Brassicacea plant family could have unique features for the regulation of GSL content in order to balance the roles of GSLs as defence compounds and as S reservoirs. During the first growing period, GSLs in leaves and root could be more involved in ameliorating S deficiency rather than plant defence. After long-term Cd exposure, we found differences between GSL patterns in leaves and root. Furthermore, we found increased total GSL levels in the stem during whole experiment which indicates that stem could serve as a GSLs storage organ under Cd stress. This paper provides new insight into the possible roles of GSL in Cd stress.

Toxic element concentrations in the bottlenose (Tursiops truncatus), striped (Stenella coeruleoalba) and Risso's (Grampus griseus) dolphins stranded in eastern Adriatic Sea.

Concentrations of cadmium (Cd), arsenic (As), mercury (Hg) and lead (Pb) were measured in muscle, liver and kidney of three cetacean species, the bottlenose (Tursiops truncatus), striped (Stenella coeruleoalba) and Risso's (Grampus griseus) dolphins from the Croatian waters of the Adriatic Sea. In all three dolphin species Cd levels decreased in tissues in the order: kidney > liver > muscle, while As and Pb decreased in the order: liver > kidney > muscle for striped and Risso's dolphins, but with the order reversed for liver and kidney in the bottlenose dolphin for Pb. Levels of Hg consistently followed the order: liver > muscle > kidney, with mean concentrations in the liver being 11-34 times higher than in the other tissues. The highest mean concentrations of trace elements were measured in Risso's dolphins at 14.9 µg/g wet weight, for Cd in the kidney, and concentrations in the liver of 2.41, 1,115 and 0.63 µg/g for As, Hg and Pb, respectively. Statistically significant differences between the three dolphin species were determined for Cd, Hg and Pb in liver tissues, for As in muscle and for Cd in kidney. Significant correlations of metals between tissues were determined in all three species. The results presented give an indication of the environmental condition with regard to the content of toxic metals along the eastern coast of the Adriatic Sea.

Lead content in multifloral honey from central Croatia over a three-year period.

Lead concentrations were analysed by atomic absorption spectrometry in multifloral honeys collected in central Croatia (Zagreb County) during a three-year period from 2009 to 2011. The mean levels of elements (µg/kg) in honey samples measured were: 90.8 in 2009, 189 in 2010 and 360 in 2011. Significant differences were observed, and Pb levels determined in 2009 were significantly lower than those in 2010 and 2011 (p < 0.05, both). In 2009 there was no concentration found above 300 µg/kg. However, in 2010 and 2011 levels exceeding 300 µg/kg were found in 28.6 % and 25 % of samples. Trace element levels of Pb determined in multifloral honey were generally higher than concentrations obtained from other geographical origins and neighbouring countries. These high concentrations of Pb may be related to the fact that the central region is becoming increasingly urban and the network of motorways is growing. Accordingly, the risk of positioning hives near zones of busy highways and railways is increasing. This underlines that particular attention should be paid to toxic Pb levels, due to their gradual increased during the study period.

Trace elements in tissues of wild carnivores and omnivores in Croatia.

The differences in metal exposure (As, Cd, Cu, Pb and Hg) in the muscle, liver and kidney tissues of brown bears (Ursus arctos), grey wolfs (Canis lupus), Eurasian lynxs (Lynx lynx), Eurasian badgers (Meles meles) and pine martens (Martes martes) from Croatia were observed. The highest mean Cd levels were found in kidney and liver of Eurasian badger (3.05 and 0.537 mg/kg). The highest Cu concentrations (mg/kg) measured in liver tissue were obtained in order: Eurasian badger (15.2) > brown bear (12.1) > pine marten (10.3) > Eurasian lynx (8.43) > grey wolf (6.44). Result presented that Eurasian badger accumulated the highest levels of elements: As, Cu and Pb in muscle; As, Cd, Cu and Pb in liver; Cd and Pb in kidney. Kidney of pine marten accumulated the highest concentrations of As, Cu and Hg. Omnivorous species observed present an important bioindicator for the accumulation of toxic elements indicating an enhanced vulnerability for response to ecological changes in forested terrain. Generally, element concentrations found in five species observed were lower in comparison to levels reported in previous studies and below levels related to toxicosis in mammals.

Concentrations of trace elements in tissues of red fox (Vulpes vulpes) and stone marten (Martes foina) from suburban and rural areas in Croatia.

Trace elements concentrations (As, Cd, Cu, Pb and Hg) were determined in the liver, kidney and muscle of 28 red fox (Vulpes vulpes) and 16 stone marten (Martes foina) from suburban and rural habitats from Croatia. Rural and suburban habitats affected Cd and Hg levels in the muscle, liver and kidney of red fox. Significant differences in metal concentrations in the muscle, liver and kidney were detected among species. Suburban stone marten accumulated the highest levels of trace elements (mg/kg w.w.): in muscle 0.019 for Hg; in liver 0.161 for Cd, 36.1 for Cu and 0.349 for Pb; in kidney 1.34 for Cd and 0.318 for Pb. Values observed were higher than those found in suburban red fox and therefore, may represent an important bioindicator for the accumulation of toxic metals in urbanized habitats.

Wild boar tissue levels of cadmium, lead and mercury in seven regions of continental Croatia.

Concentrations of cadmium, mercury and lead were analysed by atomic absorption spectrometry in the kidney and muscle of free-living wild boar (n = 169) from hunting grounds in seven counties of continental Croatia. Mean levels of metals (mg/kg) in muscle and kidney of boars ranged as follows: Cd: 0.005-0.016 and 0.866-4.58, Pb: 0.033-0.15 and 0.036-0.441, Hg: 0.004-0.012 and 0.04-0.152. In all seven regions, concentrations exceeded the permitted values (muscle and kidney mg/kg: cadmium 0.05/1; lead 0.1/0.5; mercury 0.03/0.1) in 13.6% and 71.6% of samples (muscle and kidney, respectively) for cadmium; 13.6% and 8.9% for lead; 19.5% and 2.4% for mercury. There were significant differences among the regions. Vukovar-Srijem and Virovitica-Podravina Counties were highly contaminated with cadmium, Sisak-Moslavina and Virovitica-Podravina Counties with lead and Brod-Posavina County had highest mercury concentrations. These results suggest a detailed investigation of physiological and environmental factors contributing to accumulation of metals in boars.

Survey of arsenic, cadmium, copper, mercury and lead in kidney of cattle, horse, sheep and pigs from rural areas in Croatia.

Trace element (As, Cd, Cu, Hg, Pb) concentrations were determined in the kidney of cattle, sheep, horses and pigs from rural areas of Croatia. Arsenic concentrations in kidney tissues ranged from 0.013 to 0.5 mg kg(-1). No significant differences in As kidney levels were observed among species. The highest levels of Cd and Hg were found in horses and ranged 0.029-47.4 and 0.009-0.13 mg kg(-1), respectively. The European Union maximum levels for Cd in kidney were exceeded by 92.3% of horses, 14% of cattle and 16% of sheep. The highest mean Cu levels were found in sheep and horse (8.53 and 8.45 mg kg(-1)). Mean Hg concentrations in kidney of cattle, sheep, horses and pigs were 0.051, 0.011, 0.034 and 0.094 mg kg(-1), respectively, and the highest levels of Pb were found in cattle (1.71 mg kg(-1)). Significant differences in Cd, Pb and Hg concentrations between animal species were observed.