Biomarkers detected in Chub (Leuciscus cephalus L.) to evaluate contamination of the Elbe and Vltava rivers, Czech Republic.
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Biomedical subjects
Publications and source records attributed to J Hajslová.
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Three alternative procedures were employed for the isolation of polycyclic aromatic hydrocarbons (PAHs; 15 of 16 US EPA priority pollutants and benzo[e]pyrene), their methyl-derivatives and sulphur analogues from fish tissue: (1) Soxhlet extraction, (2) batch extraction enhanced by sonication, and (3) saponification of the sample followed by re-extraction of analytes into hexane. Soxhlet extraction using hexane-acetone (1:1, v/v) was the most efficient extraction technique, with analyte recoveries in the range 70-108%. Within optimization of the clean-up step, several types of gel permeation chromatography (GPC) systems were tested: two types of polystyrene divinylbenzene copolymer gels (PSDVB), both 'soft' gel type (Bio-Beads S-X3) and 'rigid' gels type (PL gel and Envirogel) in combination with various mobile phases were compared. Bio-Beads S-X3 and mobile phase chloroform were the most appropriate for purifying of crude extracts before the final determinative step. High-performance liquid chromatography with fluorimetric detection (HPLC/FLD) was used for identification and quantification of PAHs in purified fish extracts. The uncertainties of PAHs measurements were estimated by employing two alternative approaches. Both provided similar results: the expanded uncertainties obtained for individual PAHs by the 'top-down' approach were in the range 9-53%, their values resulting from application of the 'bottom-up' approach were in the range 16-52%.
The quality of potatoes from organic and conventional farming was investigated in this study. Tubers of eight potato varieties, organically and conventionally produced at one or two geographical sites in controlled field trials, were collected in four consecutive harvests from 1996-1999. The parameters analysed included nitrate, trace elements (As, Cd, Co, Cu, Fe, Hg, Mn, Ni, Pb, Se, Zn), vitamin C, potato glycoalkaloids, as well as chlorogenic acid, polyphenol oxidase and rate of tuber enzymic browning. The results indicated lower nitrate content and higher vitamin C and chlorogenic acid content to be the parameters most consistently differentiating organically from conventionally produced potatoes. Elevated concentrations of glycoalkaloids were also observed throughout the experiments in some potato varieties grown in organic farming systems. Principal component analysis (PCA) of the analytical and other data using three PCs confirmed a good separation between the organically and conventionally produced potatoes when studied in single crop years. However, score-plots (objects) and loading-plots (variables) of pooled results from the consecutive harvests showed that between the years' changes and also variety as well as geographical variations are equally or more important factors determining the quality of potatoes than the farming system. Further studies of various marker compounds of potato quality related to the organic or conventional farming systems should be performed before unbiased information can be given to the consumers.
During 3 years of a monitoring programme, 522 samples of fresh apples, six brands of fruit purées and various types of fruit baby food prepared from these materials were analysed. Each sample was examined for the presence of 86 GC amenable pesticide residues. The reporting limits of the procedure employed for sample analyses were in the range 0.003-0.01 mg kg(-1). Pesticide residues were detected in 59.5% of the samples of fresh apples. However, maximum residue levels (European Union MRLs) were exceeded only in 1.4% of samples. The levels of residues in 'positive' fruit purées were substantially lower, overall with residues detected in 33% of samples. Fruit baby food represented the commodity with the lowest incidence of residues being detected in only 16% of samples. The 0.01 mg kg(-1) MRL was exceeded in 9% of these products. Multiple residues were found in 25% of fresh apples and in 10% of fruit purées. None of fruit baby food samples contained more than a single residue. Organophosphorus insecticides and fungicides representing phtalimides, sulphamides and dicarboximides were the most frequently found residues. To obtain more knowledge on the fate of residues during fruit baby food production, processing experiments employing apples with incurred residues (fenitrothion, phosalone and tolylfluanid) were conducted. Washing of apples did not significantly reduce the content of pesticides. Steam boiling followed by removal of peels/stems was identified as the most efficient steps in terms of residues decrease (phosalone) or complete elimination (fenitrothion and tolylfluanid).
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Agaritine (N-(gamma-L(+)-glutamyl)-4-hydroxymethyl-phenylhydrazine) was identified and quantified by high-pressure liquid chromatography and used as a marker for the occurrence of phenylhydrazine derivatives in the cultivated Agaricus bitorquis and A. garicus hortensis mushrooms. Although relatively high levels of agaritine (around 700 mg kg(-1)) could be found in freshly harvested A. bitorquis from early flushes, samples from supermarkets contained less agaritine. The content of 28 samples varied between 165 and 457 mg kg(-1), on average being 272 +/- 69 mg kg(-1). The highest amounts of agaritine were found in the skin of the cap and in the gills, the lowest being in the stem. There was no significant difference in agaritine content of the two mushroom species in our study. Pronounced reduction in agaritine content was observed during storage of mushrooms in the refrigerator or freezer, as well as during drying of the mushrooms. The degree of reduction was dependent on the length and condition of storage and was usually in the region 20-75%. No reduction in agaritine content was observed during freeze-drying. Depending on the cooking procedure, household processing of cultivated Agaricus mushrooms reduced the agaritine content to various degrees. Boiling extracted around 50% of the agaritine content into the cooking broth within 5min and degraded 20-25% of the original agaritine content of the mushrooms. Prolonged boiling, as when preparing a sauce, reduced the content in the solid mushroom further (around 10% left after 2h). Dry baking of the cultivated mushroom, a process similar to pizza baking, reduced the agaritine content by approximately 25%, whereas frying in oil or butter or deep frying resulted in a more marked reduction (35-70%). Microwave processing of the cultivated mushrooms reduced the agaritine content to one-third of the original level. Thus, the exposure to agaritine was substantially less when consuming processed Agaricus mushrooms as compared with consuming the raw mushrooms. However, it is not yet known to what extent agaritine and other phenylhydrazine derivatives occurring in the cultivated mushroom are degraded into other biologically active compounds during the cooking procedure.
Agaritine (N-(gamma-L(+)-glutamyl)-4-hydroxymethylphenylhydrazine) is a phenylhydrazine derivative found in the cultivated Agaricus mushroom which is claimed to give rise to carcinogenic products when metabolized. The stability of a synthetic sample of agaritine was tested in water and methanol. In tap water kept in open vials, agaritine was totally degraded within 48h. Since agaritine degradation was less pronounced in closed than in open vials, and slower in Milli Q water and, in particular, in Milli Q water purged with N(2), the degradation seems to be oxygen-dependent. The antioxidant dithiothreitol reduced the degradation. Four or possibly five ultraviolet-absorbing compounds were formed during degradation, but these have not yet been identified. Whereas the rate of degradation was similar at temperatures between 4 and 22 degrees C, it was quicker at an acidic than at a neutral pH. The latter observation was confirmed in experiments where agaritine was incubated in simulated gastric fluid (pH 1.2). The importance of the degradation when performing toxicological studies with agaritine is discussed.
A programmed temperature vaporizer (PTV) injection technique has been recently implemented in our laboratory. In present paper its performance is compared with other GC injection techniques commonly used in trace analysis of organic contaminants. Twenty-six pesticides representing different chemical classes were selected for the study. This group comprised compounds typically subjected to discrimination in the injection port of the gas chromatograph, e.g., polar organophosphorus pesticides and thermolabile carbamates. In the first set of experiments standards in pure solvent were injected into GC systems employing different types of injection, i.e., (i) on-column, (ii) pulsed splitless, (iii) PTV solvent split, (iv) PTV splitless, and the responses of analytes were compared. Discrimination of troublesome compounds was significantly decreased with the application of PTV solvent split injection. In the second set of experiments repetitive injections of purified wheat samples were performed, with aims to evaluate the long-term stability of responses, as well as matrix effects in different stages of system contamination for each injection technique. The tolerance of the GC system to co-injected matrix components was increased in the order: on-column<pulsed splitless<PTV solvent split technique. As regards matrix effects, these were suppressed considerably with the PTV solvent split technique in comparison with pulsed splitless injection. With the latter technique after 66 injections of wheat samples relative responses (apparent recovery) reached as much as 450% for some compounds, while with the application of PTV matrix effects did not exceed 200% under the same conditions.
A fast method of analysis for 20 representative pesticides was developed using low-pressure gas chromatography-mass spectrometry (LP-GC-MS). No special techniques for injection or detection with a common quadrupole GC-MS instrument were required to use this approach. The LP-GC-MS approach used an analytical column of 10 m x 0.53 mm I.D., 1 microm film thickness coupled with a 3 m x 0.15 mm I.D. restriction capillary at the inlet end. Thus, the conditions at the injector were similar to conventional GC methods, but sub-atmospheric pressure conditions occurred throughout the analytical column (MS provided the vacuum source). Optimal LP-GC-MS conditions were determined which achieved the fastest separation with the highest signal/noise ratio in MS detection (selected ion monitoring mode). Due to faster flow-rate, thicker film, and low pressure in the analytical column, this distinctive approach provided several benefits in the analysis of the representative pesticides versus a conventional GC-MS method, which included: (i) threefold gain in the speed of chromatographic analysis; (ii) substantially increased injection volume capacity in toluene; (iii) heightened peaks with 2 s peak widths for normal MS operation; (iv) reduced thermal degradation of thermally labile analytes, such as carbamates; and (v) due to larger sample loadability lower detection limits for compounds not limited by matrix interferences. The optimized LP-GC-MS conditions were evaluated in ruggedness testing experiments involving repetitive analyses of the 20 diverse pesticides fortified in a representative food extract (carrot), and the results were compared with the conventional GC-MS approach. The matrix interferences for the quantitation ions were worse for a few pesticides (acephate, methiocarb, dimethoate, and thiabendazole) in LP-GC-MS, but similar or better results were achieved for the 16 other analytes, and sample throughput was more than doubled with the approach.
Flash gas chromatographic (GC) analysis of 15 organophosphorus pesticides commonly occurring in food crops was performed using the Thermedics Detection EZ Flash upgrade kit installed in the oven of a HP 5890 Series II Plus gas chromatograph. The temperature program and splitless time period were the main parameters to be optimized. In the first set of experiments wheat matrix-matched standards were analyzed both by: (i) the flash GC technique (resistive heating of a 5 m capillary column), and (ii) the conventional GC technique (moderate oven temperature programming of a 30 m capillary column). Using the flash GC technique, the analysis time was reduced by a factor of more than 10 compared to the conventional GC technique. Dramatically improved detectability of analytes was achieved due to much narrower peak widths. The flash GC technique was compared with another approach to faster GC analysis employing a 5 m column and fast temperature programming with a conventional GC oven. In comparison with this alternative, in the case of flash GC significantly better retention time repeatability was observed. The other superiority of resistive heating is very rapid cooling down (i.e., equilibration to the initial conditions) which contributes to the increased sample throughput.
An effective method for the determination of seven trichothecenes-deoxynivalenol (DON), nivalenol (NIV), T-2 tetraol (T-24), fusarenon-X (FUS-X), diacetoxyscirpenol (DAS), T-2 toxin (T-2), HT-2 toxin (HT-2) in cereals is presented. Gel permeation chromatography on Bio-Beads S-X3 was used for clean-up of acetonitrile-methanol extract. GC-ECD was used for identification and quantification of trifluoroacetylated trichothecenes. The limit of quantitation for the method was in the range 40-200 micrograms/kg. Recoveries at a spiking level of 2 mg/kg ranged from 76 to 100%.
The level of agaritine was measured in fresh and canned cultivated mushroom (Agaricus bisporus) as well as in other food products containing A. bisporus, by reversed phase high performance liquid chromatography. The two fresh samples were purchased on the open market and contained 212 and 229 mg/kg, respectively. Of the 35 different trademarks of canned mushroom products studied, 25 were based on cut mushrooms and 10 on whole mushrooms. On average, whole mushrooms contained 14.9 +/- 6.7 mg agaritine per kg product whereas cut mushrooms contained 18.1 +/- 7.8 mg/kg. There was no statistically significant difference between these two values. Agaritine levels in brine were generally slightly lower than the levels detected in canned mushrooms. Thus, the level of agaritine in A. bisporus is reduced more than 10 times during the wet canning process, resulting in low levels in canned products. On a portion basis, somewhat higher amounts of agaritine may be found in some other food products (mushroom soup and pasta sauce) containing A. bisporus.
The influence of technological operations during rapeseed oil production on polycyclic aromatic hydrocarbon (PAH) concentrations in by-products, intermediate and final oils was evaluated. The decrease of light PAHs, benz(a)anthracene and benzo(a)pyrene during processing of crude oil to the deodorized product was significant at the 95% confidence interval in most batches analysed. Deodorization and alkali-refining were the steps contributing most to the PAH decrease. The relationship between PAH levels in rapeseed (and consequently in refined oil) and the duration of storage period was studied. The contamination of raw material processed a short time after harvesting was significantly higher than that of the rapeseed stored in silos for several months. Analyses of rapeseed samples, which were re-purified in the laboratory, revealed that solid particles, which contaminate rapeseed during harvesting, initial treatment, transport and storage, contributed to PAH contamination to the extent of 36% (light PAHs) to 64% (heavy PAHs) on average. Further experiments demonstrated that PAHs in re-purified rapeseed were concentrated in the cuticular layer, because they were removed well from the whole seeds by simple rinsing with organic solvent in an ultrasonic bath without losses of rapeseed oil. Alternative expressions of total PAH contamination (e.g. various PAH groups and/or differently defined B(a)P toxic equivalents) are discussed and their effect on drawing conclusions about PAH elimination rate has been demonstrated.
Glycoalkaloid levels in different cultivars collected from three regions were monitored during two years. With the exception of Karin, one of the most widely grown cultivar in the Czech Republic, none of the cultivars used for human consumption exceeded the levels considered safe. The average of total glycoalkaloids content for tested cultivars ranged from 31 to 166 mg/kg fresh weight. There were no significant differences between glycoalkaloid levels in tubers from different regions. Attention was paid to the adequate sampling procedure.
In recognition of the importance of environmental specimen banking (ESB) as an important component of the described ongoing real-time environmental and health-related monitoring programmes, a proposal for a planned ESB pilot study in the Czech Republic is presented. Selection of biomonitors, analytes, sampling techniques and sites is discussed, especially with regard to the possible harmonization with ESB already operational abroad. Availability of validated analytical techniques for determination of up to 30 elements using nuclear and spectroscopic techniques, including speciation of several metals, and of the most important organic pollutants employing various chromatographic techniques in biological and environmental samples is demonstrated.
The concentrations of persistent organochlorine contaminants including polychlorinated biphenyls (PCBs), DDT (its isomers and metabolites), hexachlorobenzene (HCB) and isomers of hexachlorocyclohexane (HCH) have been determined in human milk samples from the three regions in the Czech Republic. Results were generated by the congener-specific analyses (two-dimensional high resolution gas chromatography). The results are compared with the similar studies from other industrial countries (Norway, Great Britain, the Netherlands and Germany). Typical patterns of major PCBs contained in analyzed breast milk samples are presented. PCB No.153 was the dominant congener in all the samples. A good correlation was found between the sum of PCBs and PCB No.:153 (r = 0.99), constitutes approx. 30% of calculated PCBs content.
Basic parameters associated with practical application of gas chromatography coupled with microwave-induced plasma atomic emission spectrometric detection GC-MIP-AED in the determination of seven "indicator" polychlorinated biphenyls (PCBs) in biotic matrices were evaluated. The detection limit for chlorine (Cl-479) was found to be 0.54 pg/s. Under the conditions used for sample analysis (1 microliters of purified extract injected into the GC-MIP-AED system represented 2.5 mg of original fat), this value corresponded approximately to 0.15 mg/kg of the respective congeners in fat. The detector response was linear within the tested range of 0.5-10 ng of each injected PCB. The relative standard deviation of repeated injections for the lowest concentration level of 0.5 ng of PCB per injection ranged between 10.5 and 34.4% depending on the chlorine content of the individual analytes. The results demonstrate a high selectivity of chlorine detection. Carbon (C-496) chromatograms recorded simultaneously demonstrated the efficiency of the clean-up step used. Quantitative results (analytes at levels of 0.1-1 mg/kg) obtained with the atomic emission detector did not differ significantly from those recorded with a conventional electron-capture detector.