Migration of BPA from Food Packaging and Household Products on the Croatian Market.

BPA is a plasticizer for the production of polycarbonate plastics and epoxy resins and is widely used in the production of household goods, including food packaging. Free BPA is known to migrate from packaging to food, and its uptake has been associated with adverse health effect, particularly the disruption of endocrine activity. The presence and migration of BPA from plastic consumer products are subject to strict regulation in the EU. The aim of this study is to analyse the migration of BPA from different packaging items and household products sold on the Croatian market. To simulate real life exposure, we treated samples with a food simulant. The analytical performance was confirmed with the EU requirements. BPA levels were assessed in 61 samples by HPLC-FLD and the LOQ of the method was 0.005 mg kg-1 for the food simulant. These results showed that the levels of BPA that migrated to the food simulant were below LOQ and in accordance with the specific migration limit into food, which was defined as 0.05 mg kg-1 for all samples. None of the analysed products presented a health hazard. However, these regulations do not refer to products intended for children's use, in which BPA is banned. Furthermore, regulations require testing before putting products on the market, and previous research shows that possible BPA migration occurs due to various uses, along with a cumulative effect of exposure from even very small concentrations. Therefore, for accurate BPA consumer exposure evaluation and possible health risks, a comprehensive approach is needed.

Coffee Silver Skin-Health Safety, Nutritional Value, and Microwave Extraction of Proteins.

The aim of this research was to evaluate the health safety (concentrations of pesticide residues and heavy metals) and nutritional parameters (macro- and microminerals and crude fibre) of coffee silver skin (CS), as well to isolate proteins from this by-product using an optimised microwave extraction method. The CS by-product samples showed the highest amount of potassium, followed by calcium, magnesium, and sodium. Iron was found in the highest quantity among the microminerals, followed by copper, manganese, zinc, and chromium. The CS sample showed a large amount of fibre and a moderate quantity of proteins obtained by the optimised microwave extraction method. Four heavy metals (nickel, lead, arsenic, and cadmium) were detected, and all were under the permitted levels. Among the 265 analysed pesticides, only three showed small quantity. The results for the proteins extracted by microwave showed that the total protein concentration values ranged from 0.52 ± 0.01 mg/L to 0.77 ± 0.07 mg/L. The highest value of the concentration of total proteins (0.77 ± 0.07 mg/L) was found in the sample treated for 9 min, using a power of 200 W. Based on these results, it can be concluded that CS is a healthy and nutritionally rich nutraceutical that could be used in the production of new products in the food industry and other industries.

Chemometric Valorization of Strawberry (Fragaria x ananassa Duch.) cv. 'Albion' for the Production of Functional Juice: The Impact of Physicochemical, Toxicological, Sensory, and Bioactive Value.

Strawberries (Fragaria x ananassa Duch. cv. 'Albion') were harvested at two stages of ripeness (75% vs. 100%) and their physicochemical, sensory, toxicological, and bioactive properties were evaluated before and after processing into juice. The fresh fruits and their by-products were also evaluated. During processing into juice, the color change was higher in the fully ripe fruits, confirming the encouraging prospects for using the less ripe strawberries for processing. The analysis of heavy metals (Cu, Zn, Ni, As, Cd, Pb) was carried out, and in juice and by-product samples of 100% maturity, only Pb was higher than the MDK. Of the 566 pesticides analyzed, only cyprodinil was found in the by-products of the strawberries at 75% maturity, while pyrimethanil was detected in all samples. Fresh strawberries of both ripeness levels were rated similarly to the corresponding juices for all sensory attributes studied, indicating that sensory perception was not affected by processing. However, ripeness was found to be an important factor influencing most sensory attributes. The by-products were the materials with the highest levels of all bioactive compounds. Considering all quality parameters evaluated, the chemometric evaluation confirms the suitability of 75% ripe strawberries for processing into functional juice, which could be important for the juice industry.

Assessment of intracellular accumulation of cadmium and thallium.

The aim of the study was to develop fast and accurate method for assessment of intracellular level of cadmium (Cd) and thallium (Tl), and to establish accumulation of the metals in the cells. HepG2 cells were treated with Cd or Tl (1.0 or 10.0 mg/L; 24 h) and level of Cd or Tl was assessed. ICP-MS was applied and the method was optimized and validated. Correlation coefficient (R2) for Cd was 0.9999 with intercept 0.0732 while for Tl was 1.00009 with intercept -0.1497, and limit of detection (LOD) for Cd was 0.020 µg/L and for Tl 0.097 µg/L. Both metals, Cd and Tl, accumulate in the cells in concentration-dependent manner. However, higher uptake of Cd in comparison to Tl was observed. Cells treated with the same concentration of the metal (1.0 mg/L) accumulated 10.0% of Cd and 1.0% of Tl. Higher uptake of Cd than Tl can explain higher toxicity of Cd toward HepG2 cells. Obtained results imply to the importance of monitoring the level of metals in the cells in order to connect changes at the molecular level with exposure to specific metal.

Exposure of the Croatian adult population to acrylamide through bread and bakery products.

The aim of the present study was to determine and compare the levels of acrylamide in different types of bread and bakery products using a LC-MS/MS method, before and after the new European regulation on acrylamide reduction (Commission Regulation (EU) 2017/2158) became valid. Also, one of the aim was to estimate the average exposure to acrylamide through this food category. Of the total of 100 analysed samples, acrylamide content ranged from below the limit of quantification (LOQ) to 237 µg/kg in the period before the application of a new European Regulation, and from

Association between arsenic exposure and biomarkers of type 2 diabetes mellitus in a Croatian population: A comparative observational pilot study.

Chronic exposure to high inorganic As levels in drinking water has been related to many diseases, including type 2 diabetes mellitus (T2D). The association with low and moderate As levels, however, remains controversial and has yet not been studied in European populations. This study aimed to investigate possible association between As exposure and biomarkers of T2D in Croatian population. Observation recruited 86 adults from Eastern Croatia, where groundwater is contaminated with inorganic As, and 116 adults from Western Croatia, where As levels in drinking water are low. Both populations were divided in patient groups (T2D or prediabetes) and healthy controls. Exposure was assessed by determining total As in blood and urine and As metabolites in urine. Eastern Croatian population had a significantly higher content of As in urine than Western, whereas the opposite was true for arsenobetain. Total As and As metabolites in urine positively correlated with hemoglobin A1c (HbA1c) and negatively with albuminuria. This study provides important preliminary data on the levels of As in urine and blood and their association with biomarkers of T2D in Croatian population exposed to low or moderate levels of As through drinking water as a solid basis for further research of the pathophysiological effects of such As exposure on the status and complications of diabetes.

Stability and toxicity of differently coated selenium nanoparticles under model environmental exposure settings.

This study, motivated to fill the knowledge gap on environmental safety of selenium nanoparticles (SeNPs), provides information on the stability and environmental safety of four differently coated SeNPs rendering both positive and negative surface charges. The stability and dissolution behaviour of SeNPs were determined in an aquatic model media of different ionic strength to provide information regarding the environmental fate of SeNPs in different environmental conditions. The environmental safety of SeNPs was evaluated by acute regulatory toxicity tests using Daphina magna and Vibrio fischeri as model organisms. Agglomeration was observed for all studied SeNPs in test media with higher ionic strength caused by the disruption of surface charge leading to electrostatic instability. Toxicity of SeNPs on both aquatic species was dose-dependent and increased with exposure time. The obtained data indicated that all of the tested SeNPs could be classified as harmful to the natural bacteria V. fischeri and harmful to toxic to crustaceans D. magna, but dependent on the coating agent used for SeNPs stabilization. Although SeNPs have attracted great interest for use in biomedicine, this study demonstrated that their ecotoxicological effects should be considered during the design of new of SeNPs-based products.

Toxicity and safety study of silver and gold nanoparticles functionalized with cysteine and glutathione.

This study was designed to evaluate the nano-bio interactions between endogenous biothiols (cysteine and glutathione) with biomedically relevant, metallic nanoparticles (silver nanoparticles (AgNPs) and gold nanoparticles (AuNPs)), in order to assess the biocompatibility and fate of nanoparticles in biological systems. A systematic and comprehensive analysis revealed that the preparation of AgNPs and AuNPs in the presence of biothiols leads to nanoparticles stabilized with oxidized forms of biothiols. Their safety was tested by evaluation of cell viability, reactive oxygen species (ROS) production, apoptosis induction and DNA damage in murine fibroblast cells (L929), while ecotoxicity was tested using the aquatic model organism Daphnia magna. The toxicity of these nanoparticles was considerably lower compared to their ionic metal forms (i.e., Ag+ and Au3+). The comparison with data published on polymer-coated nanoparticles evidenced that surface modification with biothiols made them safer for the biological environment. In vitro evaluation on human cells demonstrated that the toxicity of AgNPs and AuNPs prepared in the presence of cysteine was similar to the polymer-based nanoparticles with the same core material, while the use of glutathione for nanoparticle stabilization was considerably less toxic. These results represent a significant contribution to understanding the role of biothiols on the fate and behavior of metal-based nanomaterials.

Toxicity of nanosilver and fumonisin B1 and their interactions on duckweed (Lemna minor L.).

In the environment co-contamination of several toxicants commonly occurs. However, toxicological studies usually are focused on only one toxicant. The aim of this study was to investigate toxicity of silver nanoparticles (AgNP) and mycotoxin fumonisin B1 (FB1) and their possible interactions as well as to explore tentative mechanism of their toxic effect. Duckweed (Lemna minor L.) was treated with AgNP or FB1 (at concentrations 0.5 and 1.0 mg L-1) or with their combination at same concentrations for 3 days. Both AgNP and FB1, applied individually significantly affected levels of certain nutrients, reduced growth rate and the levels of photosynthetic pigments though AgNP at a much greater extent compared to FB1. Furthermore, AgNP induced ROS generation, lipid peroxidation and increase of antioxidative enzymes activities, while FB1 induced changes only in the activities of antioxidative enzymes. Those results implicate that phytotoxicity of both AgNP and FB1 can be associated with imbalance of mineral and cell redox status. However, toxic actions of AgNp singly applied were more pronounced. Combined treatment with AgNP and FB1 produced higher degree of changes in all parameters than corresponding concentrations of AgNP or FB1 alone implying their additive effects. Additionally, higher level of FB1 found in medium, and higher level of intracellular Ag following combined treatment indicates interaction of two toxicants at the transport level/uptake in the cell which resulted in higher accumulation of Ag in duckweed cells. The latter in turn exerted higher toxicity to duckweed compared to single treatment of AgNP.

Chlorination of 5-fluorouracil: Reaction mechanism and ecotoxicity assessment of chlorinated products.

What happens to drugs in the chlorinating environment? Degradation products may vary in pharmacological profiles and in ecotoxicity potentials compared to the parent compound. This study combines synthesis, NMR spectroscopy, quantum chemical calculations, and toxicity experiments on Daphnia magna to investigate chemical fate of antineoplastic drug 5-fluorouracil (5-FU) in chlorinated environment, which is common in waste-water treatment procedures, but also endogenous in activated neutrophils. A reduction of toxicity (EC50 after 48 h is 50% higher than for the parent 5-FU) was observed after the first chlorination step, in which a chlorohydrin 5-chloro-5-fluoro-6-hydroxy-5,6-dihydrouracil was formed. Further chlorination leads to N-chlorinated intermediate, that undergoes the pyrimidine ring opening reaction. The final product, 2-chloro-2-fluoro-3,3-dihydroxypropanoic acid was obtained after the loss of the chlorinated urea fragment. This is the most potent compound in the reaction sequence, with toxicity parameter EC50, after 48 h, more than twice lower compared to the parent 5-FU. Clearly, the contact time between chlorinating species and degradation products provide different ecotoxicological properties of reaction mixtures. Interplay between experimental and theoretical procedures, to properly describe reaction pathways and provide more information on toxicity profiles, is a way forward in environmental science research.

The effect of hazardous pollutants from coal combustion activity: Phytotoxicity assessment of aqueous soil extracts.

Airborne fly ash and related hazardous particles derived from coal combustion contaminate soil and groundwater, negatively affecting ecosystems. The aim of this study was chemical and toxicological evaluation of aqueous extracts of soil collected from the vicinity of a coal-fired Plomin power plant (PPP), using Lemna (Lemna minor L.) bioassay and additional biochemical indicators - photosynthetic pigments, lipid peroxidation, antioxidative enzymes and glutathione. Topsoil samples were collected from distances of 200, 300, 400 and 800 m from the PPP in accordance with the prevailing SW wind direction. Elevated levels of polycyclic aromatic hydrocarbons (up to 15,765 ng L-1) and potentially toxic trace elements were detected in the Plomin soil extracts (PEs) in comparison to control soil extract (CE). Trace elements accumulated in L. minor were mostly in accordance with their concentrations in PEs. The results demonstrate that PEs induced significant growth inhibition and other phytotoxic effects. Those effects can be related to damage caused by increased production of reactive oxygen species and impaired antioxidant levels. The connection among the phytotoxicity, a distribution of analyzed contaminants, and distances from the PPP is clearly established.

Differences in the levels of pesticides, metals, sulphites and ochratoxin A between organically and conventionally produced wines.

Organic products are generally recognized to be healthier and safer than conventional products. However, the actual scientific data regarding the importance of organic production on particular contaminant/additive content of wines is scarce. This study aimed to evaluate contents of pesticides, metals, sulphites and ochratoxin A in organically (org) and conventionally (conv) produced wines from eleven locations in different winegrowing regions of Croatia. All wines contained significantly lower levels of residues as compared to the maximum limits (MLs) with the exception of excessive amounts of Cu and Zn in one sample. Pb and Mg were mainly significantly less represented in org wines. There were no significant differences in the content of sulphite or ochratoxin A between org and conv wines. Significantly lower total pesticide concentrations and average number of pesticides per sample were obtained in org wines. The majority of ochratoxin A positive wines were from conv wine producers.

Monitoring of selected pharmaceuticals in surface waters of Croatia.

Sulfonamides, macrolides, torasemide, fumagillin, and chloramphenicol were simultaneously analyzed in surface water samples by using solid-phase extraction (SPE) and reversed-phase (RP) liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS). In the pre-concentration and clean-up process, the pH value of samples and volume of the solvent for extraction of analytes from cartridge were optimized. Extraction recoveries were high with values in the range from 62 to 115%. Limits of quantification (LoQ) were in the range from 0.02 to 0.2 µg L-1. Repeatability of the method was evaluated at LoQ and expressed as relative standard deviation (RSD). Calculated RSDs were low with values in the range from 2.4 to 14.5%. The method was successfully applied for analysis of surface water real samples. Samples were collected along the rivers in Croatia on four sampling sites in 2012 in Danube catchment areas, 19 sampling sites in Danube and Adriatic catchment areas in 2013, and another 19 places in 2014. Altogether, 20 target compounds were analyzed in 148 water samples and detected in 31 samples in range (0.1-5.3) µg L-1 or in 20.1% of samples. The most frequent and highest concentrations were detected for macrolide antibiotics. This is the first attempt of such monitoring in surface waters in Croatia.

NITRATE IN LEAFY GREEN VEGETABLES AND ESTIMATED INTAKE.

BACKGROUND: Vegetarian diets are rich in vegetables. Green leafy vegetables are foods that contain considerable amounts of nitrate, which can have both positive and negative effects on the human body. Their potential carcinogenicity and toxicity have been proven, particularly after the reduction of nitrate to nitrite itself or just serving as a reactant with amines and/or amides in the formation of N-nitroso compounds -N-nitrosamines and other nitrogen compounds which may have high levels of nitrate. The aim of this study was to determine whether there is a significant difference, considering the location and seasonal sampling period, in the level of nitrate in certain types of green vegetables, all in order to be able to assess their intake, and possible impact on human health, especially knowing that exposure to nitrate can be potentially higher for vegetarian population group. MATERIALS AND METHODS: For this purpose, the sampling of 200 different leafy green vegetables was conducted, all of which could be found in free sale in the Republic of Croatia. The sampling was conducted during two seasonal periods - the spring and autumn period. In the springtime, lettuce (sem), spinach (pinacho), kale (kale), chard (mangel) and cabbage (brassica) were sampled, and in autumn lettuce, spinach, kale, chard and arugula. Samples were analyzed using high performance liquid chromatography (HPLC) with UV detection. RESULTS: The results from the spring sampling phase were in the range of 603 mg/kg for cabbage - 972 mg/kg for chard, and for autumn phase of 1.024 mg/kg for chard to 4.354 mg/kg for the arugula. The results showed that there were significant differences (p <0.05) for most of the samples analyzed, considering the sampling locations and time period. CONCLUSION: The results indicate that the analyzed vegetables contain significant amounts of nitrate in their composition, which represents relatively significant, but still acceptable intake into the human body.

Response of biochemical biomarkers in the aquatic crustacean Daphnia magna exposed to silver nanoparticles.

The proliferation of silver nanoparticle (AgNP) production and use owing to their antimicrobial properties justifies the need to examine the resulting environmental impacts. The discharge of biocidal nanoparticles to water bodies may pose a threat to aquatic species. This study evaluated the effects of citrate-coated AgNPs on the standardized test organism Daphnia magna Straus clone MBP996 by means of biochemical biomarker response. AgNP toxicity was compared against the toxic effect of Ag(+). The toxicity endpoints were calculated based upon measured Ag concentrations in exposure media. For AgNPs, the NOAEC and LOAEC values at 48 h were 5 and 7 µg Ag/L, respectively, while these values were 0.5 and 1 µg Ag/L, respectively, for Ag(+). The EC50 at 48 h was computed to be 12.4 ± 0.6 and 2.6 ± 0.1 µg Ag/L for AgNPs and Ag(+), respectively, with 95 % confidence intervals of 12.1-12.8 and 2.3-2.8 µg Ag/L, respectively. These results indicate significant less toxicity of AgNP compared to free Ag(+) ions. Five biomarkers were evaluated in Daphnia magna neonates after acute exposure to Ag(+) or AgNPs, including glutathione (GSH) level, reactive oxygen species (ROS) content, and catalase (CAT), acetylcholinesterase (AChE), and superoxide dismutase (SOD) activity. AgNPs induced toxicity and oxidative stress responses in D. magna neonates at tenfold higher concentrations than Ag. Biochemical methods revealed a clear increase in AChE activity, decreased ROS level, increased GSH level and CAT activity, but no significant changes in SOD activity. As Ag(+) may dissolve from AgNPs, these two types of Ag could act synergistically and produce a greater toxic response. The observed remarkably high toxicity of AgNPs (in the parts-per-billion range) to crustaceans indicates that these organisms are a vulnerable link in the aquatic food chain with regard to contamination by nanosilver. Graphical Abstract ᅟ.

Food Safety is an Important Public Health Issue: Chloramphenicol Residues Determination by Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS) in honey.

BACKGROUND: Honey is used for nutritional, medicinal and industrial purposes and antibiotic residues may harm its quality and constitute a danger to human health. The broad spectrum antibiotic chloramphenicol (CAP) was used for curative purposes in veterinary medicine, but is now forbidden in European Union (EU) because of its many serious side effects (e.g. aplastic anaemia, grey syndrome, severe bone marrow depression and hypersensitivity). The aim of this study was to facilitate analyses of the quality and safety of Croatian honey distributed to whole European Union market; an assessment that has not previously been made. SUBJECTS AND METHODS: CAP in honey was qualifying and quantifying by validated liquid chromatography tandem mass spectrometry with negative electrospray ionisation method (LC-MS/MS). The target antibiotic was separated on chromatographic column Zorbax SB C18 (150 mm x 2.1 mm, 3.5 µm) with a gradient elution using acetonitrile - 0.1% formic acid mobile phase at a flow rate of 0.3 mL/min, with column temperature 35°C for CAP and 5D-CAP as internal standard. Homogenised honey samples were diluted with acetate buffer solution and extracted on Oasis Hydrophilic-Lipophilic-Balanced (HLB) sorbents. The method was used to analyse 280 domestic honey samples collected throughout Croatia between 2005.-2013. RESULTS: Recoveries of the method for real (acacia, chestnut, linden and flower) honey samples were 102% with RSD 8.4%. The value CCα and CCß were 0.09 and 0.12 µg/kg, respectively. Results showed only three subsequent positive detections (1.1%) of CAP in honey. CONCLUSIONS: Analysed honey samples from Croatia showed good quality and safety what is the one of the main objective in consumer health policy in EU.

The determination of sulfonamides in honey by high performance liquid chromatography--mass spectrometry method (LC/MS).

The sulfonamides (SAs) are stable chemotherapeutics used against the bacterial disease affecting bees, known as American foulbrood (Bacillus larvae), so their residues could appear in the honey of treated bees. Their presence at a concentration above the limit value could be a potential danger to human health. Therefore, a simple, rapid, and reliable method for determination of 11 available SAs in honey was optimized. The samples were homogenized and cleaned with extraction on solid phase by means of Chromabond C18 end-capped cartridge followed by LC/MS analyses. A detection limit of 25 microg/kg was achieved for all analytes. The repeatability of the method was proven and the optimal parameters for temperature and pH of the mobile phase and acetic buffer, respectively, were determined. In this study, 20 samples of domestic honey were included. Six of the analyzed samples were positive, but all results were below the Croatian permissible limit value (100 microg/kg).