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Introduction Carvacrol is an essential oil extracted from oregano which can be used as a natural additive in poultry litter and could have a positive impact not only on production rates but also on the quality of poultry meat. The aim of this study was to evaluate the effect of the addition of carvacrol to litter on weight gain and the occurrence of residues in chicken tissues. Material and Methods One-day-old Ross 308 chicks were used for the study and were randomly divided into two experimental groups. For 42 days, one group was kept in a room with litter enriched with carvacrol and the second group was kept in a room with litter without carvacrol. After 42 days, the birds were sacrificed and necropsied. Carvacrol content was determined in homogenised organ tissue samples by liquid chromatography–mass spectrometry. Results Weekly weighing results showed that exposure to carvacrol in litter had no impact on chicken body weight. The analysis of plasma, muscle, liver and lung tissue after 42 days’ exposure clearly indicated that there were residues of carvacrol in the analysed matrices. Conclusion Exposure of chickens to carvacrol left residues but did not affect body weight.

Introduction Milk has been suggested to be a possible source of oestrogenically active compounds. In order to assess the health risk for milk consumers and ensure the safety of this staple part of the human diet, it is important to study the effect of xenooestrogen mixtures present in milk. This investigation used the available in vivo model to learn to what extent such compounds may be endocrine disruptors. Material and Methods The recommended immature golden hamster uterotrophic bioassay was chosen. A total of 132 animals were divided into nine groups of experimental animals and positive and negative control groups, each of 12 animals. The experimental females received ad libitum either one of five samples of raw cow’s milk from individual animals or one of four samples of pasteurised or ultra-high temperature treated cow’s milk as retail products. After 7 days, the animals were sacrificed and necropsied. Uterine weight increases were measured as the endpoint of oestrogenic activity in milk. Results The milk samples from individual cows and the retail milk samples did not show oestrogenic activity. However, in three groups, decreased uterine weights were observed. Conclusion Considering that milk supplies are beneficial to health, contamination in this food should be avoided. There is a need for further animal experiments and epidemiological studies are warranted to evaluate any causative role of milk in human endocrinological disorders.

Silver salts and azole derivatives are well known for their antimicrobial properties. Recent evidence has demonstrated also their cytotoxic and genotoxic potential toward both normal and cancer cells. Still, little is known about the action of complexes of azoles with silver(I) salts. Thus, the goal of the study was to compare the chemical, cytotoxic and antimicrobial properties of metronidazole complexes with silver(I) nitrate and silver(I) sulfate to metronidazole and pure silver(I) salts. We synthetized a novel complex, [Ag(MTZ)2]2SO4, and confirmed its chemical structure and properties using 1H and 13C NMR spectroscopy and X-Ray, IR and elemental analysis. To establish the stability of complexes [Ag(MTZ)2NO3] and [Ag(MTZ)2]2SO4, they were exposed to daylight and UV-A rays and were visually assessed. Their cytotoxicity toward human cancer cells (HepG2, Caco-2) and mice normal fibroblasts (Balb/c 3T3 clone A31) was determined by MTT, NRU, TPC and LDH assays. The micro-dilution broth method was used to evaluate their antimicrobial properties against Gram-positive and Gram-negative bacteria. A biofilm eradication study was also performed using the crystal violet method and confocal laser scanning microscopy. The photo-stability of the complexes was higher than silver(I) salts. In human cancer cells, [Ag(MTZ)2]2SO4 was more cytotoxic than Ag2SO4 and, in turn, AgNO3 was more cytotoxic than [Ag(MTZ)2NO3]. For Balb/c 3T3 cells, Ag2SO4 was more cytotoxic than [Ag(MTZ)2]2SO4, while the cytotoxicity of AgNO3 and [Ag(MTZ)2NO3] was similar. Metronidazole in the tested concentration range was non-cytotoxic for both normal and cancer cells. The complexes showed increased bioactivity against aerobic and facultative anaerobic bacteria when compared to metronidazole. For the majority of the tested bacterial strains, the silver(I) salts and complexes showed a higher antibacterial activity than MTZ; however, some bacterial strains presented the reverse effect. Our results showed that silver(I) complexes present higher photo-stability, cytotoxicity and antimicrobial activity in comparison to MTZ and, to a certain extent, to silver(I) salts.

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BackgroundThe involvement of MMP-2 and MMP-9 in the pathogenesis of various kinds of cancers including glioblastoma is well documented. The evaluation of the anticancer potential of honey bee (Apis mellifera) venom (BV) consisting of the inhibition of MMP-2 and MMP-9 secretion in a glioblastoma cell culture model was the aim of the study.Methods8-MG-BA and GAMG human primary glioblastoma cell lines vs. HT-22 mouse hippocampal neuronal cells were applied for the study. The BV dose (0.5, 1.0, 1.25, 1.5, 1.75, 2.0, 2.5, and 5.0 μg/ml) and time-dependent (24, 48, 72 h) cytotoxicity was evaluated with the tetrazolium-based colorimetric assay (MTT test). MMP-2 and MMP-9 activities in the cell culture medium under different BV concentrations were determined by gelatin zymography.ResultsA dose and time-dependent BV effect on cytotoxicity of both glioblastoma cell lines and hippocampus line was observed. The weakest, but statistically important effect was exerted by BV on HT-22 cells. The greatest cytotoxic effect of BV was observed on the 8-MG-BA line, where a statistically significant reduction in viability was observed at the lowest BV dose and the shortest incubation time. The reduction of both gelatinases secretion was observed at 8-MG-BA and GAMG lines without significant effect of HT-22 cell line.ConclusionIn vitro studies indicate that BV has both cytotoxic and inhibitory effects on the secretion of MMP-2 and MMP-9 in selected lines of glioma, suggesting anticancer properties of BV.

Quantitative, rapid, selective and sensitive methods for the determination of total arsenic (tAs) and six arsenic compounds (arsenite (As(III)), arsenate (As(V)), arsenobetaine (AsB), arsenocholine (AsC), monomethylarsonic acid (MMA), dimethylarsonic acid (DMA)) in seafood were developed. The measurement of the tAs concentration was performed using quadrupole inductively-coupled plasma mass spectrometry (ICP-MS). Microwave-assisted extraction was used for the isolation of arsenic species. The separation and quantification of analysed compounds were performed by ion-exchange chromatography coupled with ICP-MS in one chromatographic run using ammonium carbonate-based buffers, which has little effect on ICP-MS sensitivity compared to commonly used phosphate buffers. The results of validation and proficiency tests confirmed the reliability, robustness, and applicability of the developed procedures to various types of matrices. The proposed methods are relatively simple, time- and cost-efficient, therefore could be used to routinely analyse tAs content and arsenic species in different types of seafood at trace and ultra-trace levels.

Introduction The use of growth promoters in animal husbandry to increase weight gain and efficiency of feed conversion into muscle has been banned in the European Union since 1988, and under Directive 96/23/EC, surveillance for anabolic steroid hormones is obligatory. The hormones present in animal tissues may be of endogenous origin or may result from illegal administration. Steps have been taken to determine selected steroids in the form of esters in the alternative matrix of animal hair. Their detection in biological material is direct proof of the illegal use of anabolics. Material and Methods The procedure for the determination of steroid esters in animal hair, based on digestion, extraction, purification, and liquid chromatography with tandem mass spectrometry was validated under the current regulations. In total, 348 samples of animal hair were examined using this method. Results Good recoveries and precision values (RSD) were obtained during validation. Decision limits (CCα) and detection capabilities (CCβ) were in the ranges of 2.57–4.18 μg kg−1 and 4.38–7.12 μg kg−1, respectively. The method met the criteria for confirmation techniques with respect to Commission Decision 2002/657/EC. Conclusion Testing for steroid esters in animal hair was introduced into the National Residue Control Programme in 2017. Steroid esters were not found in any hair samples above the CCα, which indicates that illegal use of anabolics was not confirmed.

For the detection of 19 steroid hormones in bovine muscle, a fast and sensitive liquid chromatography with electrospray ionization tandem mass spectrometry method was developed using both positive and negative ionization mode. Chromatographic separation on Poroshell 120-EC C18 column was achieved in less than 10 min using isocratic elution of mobile phase of acetonitrile/methanol/water. The compounds were extracted from muscle tissue using ethyl acetate and quick, easy, cheap, effective, rugged, and safe technique. The purification of the obtained extract was performed by dispersive solid-phase extraction with sorbents C18, primary secondary amine and magnesium sulphate. The method was validated in accordance with the Commission Decision 2002/657/EC. For all steroids tested good recoveries were obtained (from 51.2 to 121.4%) in the concentration range from decision limits until 5 µg/kg. The values of decision limits and the detection capabilities for individual compounds were in the range 0.10-0.48 and 0.17-0.95 µg/kg, respectively. The method was characterized by satisfactory linearity for most compounds (correlation coefficients > 0.99) and the reproducibility was lower than 35%. The elaborated procedure has met the criteria for confirmatory methods and is currently used in the official control of hormones.

Vilazodone (VLZ) is a drug approved for the treatment of major depressive disorder in humans but no data are available for dogs. The present study aimed to evaluate the pharmacokinetics of a single oral 40 mg dose of VLZ in healthy Labrador dogs (n = 6) in fasted and fed conditions. Dogs were randomly divided in two (n = 3) groups in a cross-over study design (2 × 2). Group I was administered with VLZ at 40 mg/dog after fasting over-night. Group II was fed prior to and after administration of the same dose. A two-week wash-out period was observed. Plasma samples collected underwent LC-MS/MS analysis. VLZ concentrations were quantified in dogs' plasma in two different windows of time: 30 min to 10 h for the fasted group and 4 h to 35 h for the fed group. The values for t1/2λz were statistically different between the groups (fed, 4.6 ± 1.1 h vs fasted, 1.7 ± 0.2 h). Tmax drastically changed between the groups (fed, 10 h vs fasted, 1.5 h), while Cmax did not significantly vary (fed, 39.4 ± 5.6 ng/mL vs fasted, 38.7 ± 4.8 ng/mL). The AUC value was always statistically higher in the fed group. As a result, the average relative oral fasted bioavailability of VLZ was low, 28.8 ± 6.1%. In conclusion, feeding can affect the pharmacokinetics of VLZ in the dog.

Current work presents a modified QuEChERS method for the determination of 207 pesticide residues in honey by LC–MS/MS and GC–MS/MS. Acetate buffered acetonitrile extraction with Z-Sep+ and PSA dispersive-SPE clean-up were used for sample preparation. Optimised conditions allows determination of neonicotinoids as well as other insecticides, fungicides, herbicides, acaricides, growth regulators and veterinary drugs in honey samples. Validated method enable sensitive analysis at least at concentrations from 0.001, 0.005 or 0.01 mg/kg for 45%, 41% and 14% of pesticides, respectively. Method was utilised for the analysis of 155 honey samples from Poland during 2015–2017. Residues of 21 pesticides were determined in honey. Cyano-substituted neonicotinoids (acetamiprid, thiacloprid) were quantified in 77% of samples and were the most frequently detected pesticides. Concentrations of acetamiprid was from 0.001 to 0.13 mg/kg whilst thiacloprid from 0.001 to 0.2 mg/kg. Fungicides were determined in 50% and amitraz metabolites in 35% of honey samples.

Introduction: Quercetin is a polyphenolic flavonoid which has been used in traditional Chinese medicine as a natural therapeutic agent with a broad spectrum of activities (antioxidant, anticancer, neuroprotective, anti-inflammatory, antiviral and antibacterial). The aim of this study was to develop and validate a rapid and simple ultra-high-performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) method for the determination of quercetin in milk. Material and Methods: Sample preparation was based on a liquid-liquid extraction with 0.5% formic acid in acetonitrile. The chromatographic separation was performed on a ZORBAX SB-C18 column with methanol and 0.5% formic acid as a mobile phase. Results: The procedure was successfully validated. The mean recovery of the analyte was 98%, with the corresponding intra- and inter-day variation less than 10% and 15%, respectively, and the repeatability and reproducibility were in the range of 3%–7.2% and 6.1%–12%, respectively. The lowest level of quantification was 1.0 μg/kg. Conclusion: The proposed method was successfully applied in evaluating the pharmacokinetics of quercetin in milk obtained from dairy cows with clinical mastitis after intramammary administration.

Resorcylic Acid Lactones, including zeranol, anabolics listed in the group A4 of Directive 96/23/EC, are banned in Europe for use in animals since 1985. Zeranol, after administration to animals, is metabolized to taleranol and zearalanone. It can also naturally occur in the urine due to conversion of zearalenone that may be present in animal feed. In 2010-2017, in Poland, 7746 animal samples were tested for zeranol residues within the official monitoring program. In 13, zeranol was detected after screening. Re-examinations confirmed resorcylic acid lactones in six samples. The recommendations state that only the presence of zeranol and/or taleranol gives the basis for non-compliance. Confirmation should cover the entire profile of six resorcylic acid lactones. In case of detection, the relationship ratio should be verified. Following the proposed criteria, it could be concluded that zeranol detected in urine samples in Poland originated from contamination of feed with mycotoxin, not from illegal use.

The article reports a case of acute copper sulfate (CuSO4) poisoning in 2-day-old turkeys. The first incidences were noticed 2 h after placing the animals in the poultry house, where turkeys were provided with wood shaving bedding. Post-mortem examination showed wood shavings present in gastric and intestinal contents and numerous lesions in the alimentary tract: dark green color of the koilin lining, and surface erosions, deep ulceration, and severe congestion of the gizzard. The copper (Cu) concentration in samples of the liver, kidney, heart, gizzard content, gizzard muscle, gizzard lining, and wood shavings from the bedding was determined. Our results showed that the level of Cu in the livers of affected birds was more than sevenfold higher than the recommended value and the Cu content in bedding material was above the value considered to be toxic to turkeys. Our investigation identified the bedding disinfected using a CuSO4 solution as a possible cause of turkey fatalities.

Introduction: In the European Union, the use of thyreostatic drugs for fattening slaughter animals has been banned since 1981 under Council Directive 81/602/EEC. For protection of consumer health against unwanted residues and in compliance with Directive 96/23, each EU country must monitor thyreostats in samples of animal origin. This paper presents the results of research on thyreostatic residues carried out in Poland in 2011–2017. Material and Methods: The material for testing was urine (n = 3,491), drinking water (n = 127), and muscle samples (n = 349) officially collected by Veterinary Sanitary Inspectors in slaughterhouses and farms throughout the country in accordance with the national residue control plan. The samples were examined for the presence of tapazole, thiouracil, methylthiouracil, propylthiouracil, and phenylthiouracil using liquid chromatography tandem mass spectrometry through an accredited method. Results: In four bovine and three porcine urine samples, the permissible thiouracil concentration was exceeded. In one sample of porcine urine, methyl- and propylthiouracil were found. The presence of thiouracil and its derivatives in urine samples is most likely due to feeding animals diet containing cruciferous plants. Conclusions: The results of research indicate that thyreostats are not used for anabolic purposes in slaughter animals in Poland.

Many persistent organic pollutants (POPs) exhibit endocrine disrupting activity but studies on some POPs, e.g., polychlorinated naphthalenes (PCNs), are very scarce. The present study investigates the (anti)estrogenic and (anti)androgenic activities of 1,2,3,5,6,7-hexachloronaphthalane (PCN67) and 1,3,5,8-tetrachloronaphthalene (PCN43) using the yeast estrogen and androgen reporter bioassays. Among the tested substances, antiestrogenic response was only shown by PCN67. The strongest inhibition of estrogenic activity (up to 17.4%) was observed in the low concentration ranges (5 pM - 0.5 nM) in the presence of 1.5 nM 17β-estradiol. Both tested compounds showed partial estrogenic activity with a hormetic-type response. However, both studied chemicals showed strong antiandrogenic effects: their potency in the presence of 100 nM 17β-testosterone for PCN43 (IC50 = 2.59 μM) and PCN67 (IC50 = 3.14 μM) was approximately twice that of the reference antiandrogen flutamide (IC50 = 6.14 μM). It cannot be excluded that exposure to PCNs, together with other endocrine disrupting chemicals (EDCs), may contribute to the deregulation of sex steroid hormone signaling.

A liquid chromatography-tandem mass spectrometry method was developed for simultaneous determination of deoxynivalenol (DON), 3-acetyldeoxynivalenol (3Ac-DON), 15-acetyldeoxynivalenol (15Ac-DON), DON-3-glucoside (DON-3Glc) nivalenol and fusarenone-X in feedstuffs. Different techniques of sample preparation were tested: solid-liquid-extraction, QuEChERS, solid phase extraction with OASIS HLB columns or immunoaffinity columns and a Mycosep 225 Trich column. None of the six immunoaffinity columns tested showed cross-reactivity to all of the mycotoxins. Surprisingly, the results show that if the immunoaffinity columns bound 3Ac-DON, then they did not bind 15Ac-DON. The most efficient sample preparation was achieved with a Mycosep 225 Trich column clean-up. The chromatography was optimised to obtain full separation of all analytes (including 3Ac-DON and 15Ac-DON isomeric form). The validation results show the relative standard deviations for repeatability and reproducibility varied from 4% to 24%. The apparent recovery ranged between 92% and 97%, and the limit of quantification described a 1.30 to 50 &micro, g/kg range. The method trueness was satisfactory, as assessed by a proficiency test and analysis of reference material. A total of 99 feed samples were analysed by the developed method, revealing the presence of DON and DON-3Glc in 85% and 86% of examined animal feeds, respectively at concentrations between 1.70 and 1709 &micro, g/kg. The ratios DON-3Glc to DON in the surveyed feedstuffs were from a low of 3% to high of 59%.

Anabolic hormones, which cause muscle growth, have been banned for anabolic purposes in animal husbandry in Europe since the 1980s. Control of hormones from the list of Annex I to Directive 96/23/EC is mandatory in the European Union. The presence of hormones in samples of animal origin may be due to their endogeneous nature or illegal use. One way to distinguish their origin is to study hormones, particularly steroids in the form of ester derivatives. In the body synthetic hormone esters could be only exogenous therefore their detection in animal tissues is the undisputed evidence of illegal administration. The analytical procedure involves the extraction of esters from serum with organic solvents, derivatisation with methoxyamine and detection by liquid chromatography tandem mass spectrometry. The method was approved in accordance with the applicable legislative criteria and its effectiveness was verified in the proficiency test. The research material consisted of bovine serum samples officially taken. During the validation good apparent recovery, precision, decision limits and detection capabilities in the range 0.006–0.012 µg L−1 and 0.010–0.020 µg L−1 respectively were obtained. The developed method met the criteria for confirmation set out in Commission Decision 2002/657/EC. Since the inclusion of serum in 2018 for testing for testosterone esters in the National Residue Control Program, 130 samples have been examined. In none of the serum samples, esters above the decision limits were found. The control of animals and food of animal origin for hormone esters will be continued to ensure the health and safety of consumers.

Two novel silver(I) complexes of the biologically active ligand miconazole in the form of Ag(MCZ)2X (MCZ = 1-[2-(2,4-dichlorobenzyloxy)-2-(2,4-dichlorophenyl)ethyl]-1H-imidazole], X = NO3&minus, (1), ClO4&minus, (2)) were synthesized and fully characterized. The complexes were obtained by reactions of Ag(I) salts with miconazole (MCZ). Silver(I) complexes were characterized by elemental analysis, 1H-NMR and infrared (IR) spectroscopy, electrospray ionization (ESI)-MS spectrometry, and X-ray-crystallography. This work also presents a cytotoxicity study of the silver(I) complexes of miconazole and appropriate silver(I) salts using Balb/c 3T3 and HepG2 cell lines. The cytotoxicity of the compounds was assessed based on four biochemical endpoints: lysosomal activity (neutral red uptake (NRU) assay), mitochondrial activity (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay), total protein content (TPC assay), and cellular membrane integrity (lactate dehydrogenase (LDH) assay). The cancer HepG2 cells were more sensitive to the complexes tested, and the most affected endpoint was cellular membrane damage compared to Balb/c 3T3 fibroblasts. Moreover, study complexes inhibited the growth of cancer cells at submicromolecular concentrations (0.26&ndash, 0.47 &mu, M) lower than that required for the anticancer agent, cisplatin, in MTT, NRU, and TPC assays. Both complexes were characterized by higher toxicity to human cancer cells (HepG2) than silver(I) salts and the free ligand. Combination of Ag(I) salts with miconazole is associated with the marked improvement of cytotoxic activities that can be considered as the significant point in the construction of a new generation of antineoplastic agents.

Introduction The major difficulty in analysis of nitrofurans in feed, feed water, and food of animal origin is that nitrofurans have low molecular weights and fast metabolism. The principal goal of this study was to prepare a procedure for the determination of nitrofurans and their metabolites by a single method in different types of feed, feed water, and food of animal origin. Material and Methods Two-gram samples were subjected to hydrolysis and derivatisation processes by addition of hydrochloric acid and 2-nitrobenzaldehyde. After incubation the sample was purified by solid phase extraction technique. Nitrofurans were analysed using ultra-high-pressure liquid chromatography-MS/MS (UHPLC-MS/MS). Results The results of validation fulfil the requirement of the confirmatory criteria according to the European Commission Decision 2002/657/EC regarding apparent recoveries (88.9%–107.3%), repeatability (2.9%–9.4%) and within-laboratory reproducibility (4.4%–10.7%). Conclusion The method can be successfully applied to monitor nitrofurans and their metabolites in different matrices.

Beeswax is a valuable honeybee product, which finds applications in the food and pharmaceutical industries, as well as in cosmetic production. However, some substances used in apiculture, like tetracyclines, can be delivered to hives where they cause contamination of honeybee products such as beeswax. Tetracyclines are commonly used for the treatment of American and European foulbrood diseases, but in the European Union their usage by beekeepers is forbidden. Thus, a sensitive method for the analysis of tetracyclines in beeswax is an important analytical tool. A new liquid chromatography with tandem mass spectrometry method for the analysis of tetracyclines including oxytetracycline, 4-epioxytetracycline, tetracycline, 4-epitetracycline, chlorotetracycline, 4-epichlorotetracycline, and doxycycline in beeswax was developed. The method involved dilution of beeswax in n-hexane after a melting step, liquid-liquid extraction with oxalic acid and clean-up using a weak cation exchange phase. Satisfactory separation was performed on an octadecyl column with 0.1% formic acid and acetonitrile in a total run time of 5 min. The application of this method was evaluated by the analysis of real beeswax samples. The presence of oxytetracycline was confirmed in 5 out of 48 tested beeswax samples, which shows the method can be successfully used to determine the tetracyclines in beeswax.

Introduction Amitraz is a formamide exhibiting both acaricidal and insecticidal activity and is frequently used by beekeepers to protect honeybee colonies against Varroa destructor mites. The aim of this apiary trial was to evaluate the impact of honeybee colony fumigation with amitraz on the level of contamination of honey stored in combs. Material and Methods Experimental colonies were fumigated four times every four days with one tablet of Apiwarol per treatment. Honey was sampled from combs of brood chambers and combs of supers one day after each amitraz application and from harvested honey. Amitraz marker residues (as a total of amitraz and metabolites containing parts of molecules with properties specific to the 2,4-DMA group, expressed as amitraz) were evaluated in honey. Results All analysed samples were contaminated with amitraz metabolites. 2,4-DMA and DMPF were the most frequently determined compounds. The average concentration of amitraz marker residue in honey from groups where a smouldering tablet was located directly in beehives was significantly higher than that of residue in honey from groups with indirect smoke generation. No significant effect on the honey contamination deriving from the place where it was exposed to smoke (combs of brood chambers and supers) was noted. Amitraz marker residues exceeded the MRL in 10% of honey samples from combs. Conclusion Fumigation of beehives with amitraz results in contamination of honey stored in combs.

Thiouracil (TU) is a representative of a group of thyreostatics, orally active drugs that can be used to increase the weight of cattle before slaughter. The use of thyreostats in animal production has been banned in the European Union since 1981. Systematic detection of low concentrations of thiouracil in the urine of livestock is acknowledged to be of endogenous origin due to the use of Brassicaceae plants in the animal diet. The purpose of the study was to determine the effect of diet enriched with rapeseed meal on endogenous thiouracil contents in urine of calves. Two groups of calves of different age were subjected to a rapeseed-supplemented diet. First group (n = 6, 6 months) received a maximum authorized content of rapeseed meal (30%) in diet, second (n = 2, 2 months) after adaptation to solid food and rapeseed, has been subjected to a diet exceeding the maximum recommended dose of rapeseed of 10%. During the experiments, samples of urine and faeces were collected and analyzed by using the accredited LC-MS/MS method. The diet with allowed content of rapeseed did not influenced on concentration of endogenous thiouracil in calves' urine (CCα - 3.11 μg/L), while exceeding the permitted dose caused a significant increase in thiouracil in urine of younger animals (1.55-20.95 μg/L) and there may be a reason for non-compliant results. The higher levels of endogenous thiouracil observed in young calves results from exceeding the recommended dose of rapeseed in diet as well as from intensive feeding of animals during growth.

A method based on ultra-high performance liquid chromatography was developed and validated to detect six thyreostatic compounds: tapazole, thiouracil, methylthiouracil, dimethylthiouracil, propylthiouracil, and phenylthiouracil in faeces of bovine. Thyreostats were extracted from the matrix with a mixture of methanol and buffer (pH = 8). Next step was derivatization of analytes with 3-iodobenzylbromide. The liquid chromatographic separation of derivatives was obtained on a SB-C18 column (50 × 2.1 mm; 1.8 μm, Agilent) with gradient elution using a mobile phase consisting of acetonitrile/0.1% acetic acid within 7.5 min. The analysis was performed on a Shimadzu NEXERA X2 ultra-high performance liquid chromatograph with triple quadrupole MS 8050 instrument operating in positive electrospray ionization mode. Depending on the target compound, two or three diagnostic signals (selected reaction monitoring transitions) were monitored. The procedure was validated according to the Commission Decision 2002/657/EC. Recovery and repeatability met the performance criteria specified by this document for banned compounds. The recovery ranged from 97.5 to 110.5%, and repeatability did not exceed 14.1%. Decision limits and detection capabilities were below 10 μg/kg. The highest decision limits and detection capabilities concentrations were observed for phenylthiouracil of 3.48 and 6.96 μg/kg, respectively.

Sudan I is a carcinogenic industrial azo-dye, forbidden for use in food. However, it has been detected in food on several occasions, such as in paprika, used in animal husbandry to enhance egg yolk colour. Therefore, an animal experiment was designed to simulate the transfer of Sudan I to eggs after its unintentional administration to laying hens. A group of laying hens (n = 18) received feed contaminated with Sudan I at the raising concentrations: 0.45 mg/kg, 4.97 mg/kg and 42.1 mg/kg. Residues of Sudan I were detected in egg yolks (0.29 ± 0.03 µg/kg, mean ± SD) only after the administration of the feed contaminated with the dye at the highest concentration. The determined concentrations were much lower than expected based on the compound’s lipophilicity. In conclusion, the transfer of Sudan I to eggs was limited and strongly dependent on its concentration in feed.

Current work presents developed and validated miniaturized method for residue analysis of 261 pesticides and their metabolites as well as 6 congeners of non-dioxin like polychlorinated biphenyls (ndl-PCB) in a very low mass beebread sample. Sample preparation is based on modified QuEChERS protocol with all steps miniaturized to enable multiresidue analysis of sample with extremely low weight. Sample of beebread (0.3 g) was extracted with 1 mL of acetonitrile containing 5% formic acid and ammonium formate salt were added, then extract was subjected to clean-up by freezing and two-step dispersive solid phase extraction (dSPE) with a Supel QuE Verde sorbents (Supelclean ENVI-Carb Y; Supelclean PSA; Z-Sep+; magnesium sulfate). After 1st step dSPE a portion of extract was analyzed by liquid chromatography tandem mass spectrometry (LC-MS/MS) for 200 pesticide residues. Remaining extract was subjected to 2nd step dSPE clean-up by another Supel QuE Verde and then after concentration and solvent exchange it was analyzed by gas chromatography tandem mass spectrometry (GC-MS/MS) for another 61 pesticide and 6 ndl-PCB residues. Method enables determination of residues of 101 insecticides, 72 herbicides, 67 fungicides, 10 acaricides, 6 growth regulators, 5 veterinary drugs and 6 ndl-PCB's. Particular attention was paid to the pesticides being active substances of plant protection products recommended for the protection of winter oilseed rape and apple orchards which during their blooming periods are one of the most attractive sources of food for pollinators and could serve as representatives of other economically important crops. Method was validated according to the Guidance document SANTE/12682/2019 at six concentration levels from 0.001 to 0.5 mg kg−1. The analysis of beebread samples spiked at the level of 0.01 mg kg−1showed mean recovery (trueness) value of about 98% and RSDr (precision) below 20%. The small weight of the sample did not adversely affect the limits of quantification and 75% of analytes could be quantified at least at concentration of 0.005 mg kg−1. Developed mini-method was tested in the analysis of beebread samples, each extracted from individual cell of honeycomb. It is the first time when analyses at single comb cell level were possible.

Bee pollination is economically and ecologically vital and recent declines in bee populations are therefore a concern. One possible cause of bee declines is pesticide use. Bumblebees exposed to imidacloprid, a neonicotinoid pesticide, have been shown to be less efficient foragers and collect less pollen on foraging trips than unexposed bees. We investigated whether bumblebees (Bombus impatiens) chronically exposed to imidacloprid at field-realistic levels of 2.6 and 10 ppb showed learning deficits that could affect foraging. Bumblebees were tested for their ability to associate flower colour with reward value in a simulated foraging environment. Bumblebees completed 10 foraging trips in which they collected sucrose solution from artificial flowers that varied in sucrose concentration. The reward quality of each artificial flower was predicted by corolla colour. Unexposed bumblebees acquired a preference for feeding on the most rewarding flower colour on the second foraging trip, while bumblebees exposed at 2.6 and 10 ppb did not until their third and fifth trip, respectively. The delay in preference acquisition in exposed bumblebees may be due to reduced flower sampling and shorter foraging trips. These results show that bumblebees exposed to imidacloprid are slow to learn the reward value of flowers and this may explain previously observed foraging inefficiencies associated with pesticide exposure.

A series of novel indole-azolidinone hybrids has been synthesized via Knoevenagel reaction of 5-fluoro-3-formyl-1H-indole-2-carboxylic acid methyl ester and some azolidinones differing in heteroatoms in positions 1, 2 and 4. Their anticancer activity in vitro was screened towards MCF-7 (breast cancer), HCT116 (colon cancer), HepG2 (hepatoma), HeLa (cervical cancer), A549 (lung cancer), WM793 (melanoma) and THP-1 (leukemia) cell lines, and a highly active 5-fluoro-3-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)-1H-indole-2-carboxylic acid methyl ester (3a) was identified and subjected to in-depth investigation of cytotoxicity mechanisms. This compound was found to possess the highest cytotoxic action towards tumor cells comparing with the action of other derivatives (1, 3b, 3c, 3d, 3e). Compound 3a exhibited toxicity toward MCF-7, HCT116, and A549, HepG2 cancer cells, while the non-malignant cells (human keratinocytes of HaCaT line and murine embryonic fibroblasts of Balb/c 3T3 line) possessed moderate sensitivity to it. The compound 3a induced apoptosis in studied tumor cells via caspase 3-, PARP1-, and Bax-dependent mechanisms; however, it did not affect the G1/S transition in HepG2 cells. The compound 3a impaired nuclear DNA in HepG2, HCT116, and MCF-7 cells without intercalating this biomolecule, but much less DNA damage events were induced by 3a in normal Balb/c 3T3 fibroblasts compared with HepG2 carcinoma cells. Thus, 5-fluoro-3-(4-oxo-2-thioxothiazolidin-5-ylidenemethyl)-1H-indole-2-carboxylic acid methyl ester 3a was shown to trigger DNA damage and induce apoptosis of human tumor cells and it might be considered as an anticancer agent perspective for in-depth studies.

Regardless of whether antimicrobial drugs are administered to laying hens legally or illegally, residues of these drugs may be present in the eggs. Even if the eggs are not intended for human consumption, byproducts/biowaste, such as eggshells, may contain residues of the drugs used, which may pose a risk to human health and the environment. In the presented research, 2 different groups of laying hens received enrofloxacin (10 mg/kg body weight) and lincomycin (20 mg/kg body weight) once daily for 5 d. Eggs were collected daily and the concentration of enrofloxacin, its metabolite ciprofloxacin, and lincomycin residue in the eggshells, whole eggs, egg yolks, and egg whites were determined by ultra-high-performance liquid chromatography-tandem mass spectrometry. This study demonstrates the transfer of enrofloxacin, ciprofloxacin, and lincomycin into the eggshells and provides evidence for the distribution into the eggshells after administration of these drugs to laying hens. The enrofloxacin residues were detected in the eggshell for 10 d after cessation of treatment, ciprofloxacin and lincomycin were rapidly eliminated and 2 d after finish drugs administration they were no longer detected in the eggshell.

Alternariol (AOH) is a toxic metabolite of phytopathogenic fungi of the Alternaria spp. and important contaminant of agricultural commodities. According to the recent studies, AOH has a potential to modulate the endocrine system of humans and animals. In the view of these reports, our study addressed the effects of AOH on human estrogen receptor (hERα) and androgen receptor (hAR) signaling with the use of the yeast estrogen and androgen reporter bioassays. Our results show that, apart from a weak estrogenic response, AOH induces full androgenic response of the bioassay with the EC50 of 269.4μM. The androgenic potency of AOH relative to testosterone (T) is 0.046%. Moreover, in the presence of T, AOH at 5μM acts as a weak antiandrogen, whereas at higher concentrations AOH sum up with the androgenic activity of T in a dose-dependent manner, suggesting additive effect. To our knowledge it is the first report of the androgenic potency of natural, nonsteroidal substance and may have the impact on the direction of the further studies. Further research is warranted to clarify the role of AOH in disruption of AR signaling in humans and animals.

Introduction: The paper presents the method of simultaneous determination of 10 illegal azo dyes in feed by ultra-high performance liquid chromatography coupled with tandem mass spectrometry technique. Material and Methods: The dyes were extracted with hexane, evaporated to dryness, and analysed. Separation was achieved in 7 min in a gradient elution using acetonitrile (A) and 0.1% formic acid (B) as a mobile phase. Results: The validation results showed the repeatability of the method, which was evaluated at three levels (50, 500, and 5,000 μg/kg). All the matrix calibration curves for the working ranges were linear (R2 0.9904 to 1.0), the repeatability was between 2.1% and 24%, and recoveries ranged from 77.9% to 120%. The LOD and LOQ were at 1-2 and 5-10 μg/kg for different dyes, respectively. Furthermore, the method was applied in the homogeneity tests of the in-house prepared feed containing Sudan I at the levels of 0.5, 5, and 50 mg/kg. Conclusions: A sensitive, selective, and fast multiresidue method was successfully developed and validated. Its robustness was confirmed by the analysis of an experimental feed containing Sudan I.

Introduction: The main problem in determination of chloramphenicol in food of animal origin is a large number of matrices. The main target of this study was to create a method for determination and confirmation of chloramphenicol in products and food of animal origin. Material and Methods: Each 5 g matrix sample was mixed with 5 mL of water and 10 mL of acetonitrile/ethyl acetate, homogenised, and centrifuged. The organic layer was evaporated and redissolved in 6 mL of 4% NaCl. The extract was cleaned up by SPE technique. Chloramphenicol was analysed by LC-MS/MS in electrospray mode. Results: The procedure was validated according to the Commission Decision No. 2002/657/EC. The apparent recoveries were in the range of 92.1% to 107.1% with a repeatability less than 11.0% (4.4%-11.0%) and within-laboratory reproducibility below 13.6% (4.7%-13.6%). Conclusion: The method was successfully validated and proved to be efficient, precise, and useful for quantification of chloramphenicol in more than 20 different matrices.

W pracy przedstawiono porównanie aktywności estrogennej 30 produktów mleczarskich ogólnie dostępnych na polskim rynku. Próbkami wybranymi do badania były odtłuszczone, półtłuste i tłuste mleko krowie, mleko kozie, ser pleśniowy, jogurt, twaróg, śmietana i masło. Aktywność estrogenną w próbkach badano z użyciem testu in vitro opartego na transgenicznych komórkach drożdży po hydrolizie enzymatycznej i ekstrakcji dSPE. Aktywność estrogenną stwierdzono w 56, 7% próbek. Stwierdzono dodatnią korelację pomiędzy zawartością tłuszczu w badanych próbkach a zmierzoną aktywnością estrogenną. Najwyższą aktywność estrogenną stwierdzono dla próbek masła, najniższą dla mleka odtłuszczonego. Uzyskane wyniki pozwalają sądzić, że mleko i jego produkty mogą być ważnym źródłem estrogenów, zwłaszcza dla grup najbardziej wrażliwych.

Introduction: In the European Union the use of steroid growth promoters is prohibited under Council Directive 96/22/EC. For effective control of illegal use of natural steroids, highly sensitive analytical methods are required, because sex hormones can be present in very low concentrations in biological samples. The aim of the study was to develop a confirmatory method for the detection of testosterone in bovine serum at ppt level. Material and Methods: 17β-testosterone and internal standards of 17β-testosterone-d2 were extracted from serum samples with a mixture of tert-butyl methyl ether/petroleum ether and were directly analysed by an LC/MS/MS on QTRAP 5500 instrument with a TurboIon-Spray source operating in a positive ionisation mode. Chromatographic separation was achieved on the analytical column Inertsil® ODS-3 with an isocratic elution using mobile phase consisting of acetonitrile, methanol, and water. Method validation has been carried out in accordance with the Commission Decision 2002/657/EC. Results: The method was characterised by good recovery (82%) and precision (R.S.D 17 %). Decision limit (CCα) and detection capability (CCβ) was 0.05 μg L−1 and 0.09 μg L−1 respectively. The method met the criteria set out in Commission Decision 2002/657/EC for the purpose of confirmation in terms of retention time and ion ratio in the whole range of its application. Conclusions: The developed method is specific and sensitive, suitable for measuring the natural level of testosterone in blood of cattle and for use in routine control programme for the detection of this hormone in bovine serum.

Introduction: Studies of anabolic hormone residues in the tissues of slaughter animals have been carried out in Poland for more than 25 years. During the period of 2011 to 2015, a total of 35 387 samples from different animal species were tested in the National Residue Control Programme for the presence of residues of compounds that cause hormonal effects, as listed in Annex 1 of Directive 96/23/EC. Material and Methods: The research was conducted in the National Reference Laboratory and eight regional laboratories in departments of veterinary hygiene located throughout the country. Urine, muscle tissue, serum, kidney fat, and drinking water were the targeted matrices. Test methods based on instrumental techniques such as gas and liquid chromatography coupled with mass spectrometry were applied, as well as enzyme-linked immunosorbent assays (ELISA). Results: The concentration of detected hormones exceeded the decision limits in 30 samples, the consequence of which was 41 non-compliances with current applicable criteria. The hormones found present pseudo-endogenous (nortestosterone and boldenone) only, while synthetic hormones were not identified. Conclusion: The non-compliant findings constitute a small percentage (0.085%) of the five-year analysis compilation. On this basis the related food produced in Poland can be accepted as safe for human consumption with regard to the hormone residues tested.

The presence of antibiotic residues in pig tissues requires a search for new methods for their antemortem detection. To find an alternative for postmortem pig carcass analysis, an oral fluid was tested. To prove the suitability of oral fluid for the detection of antibiotics administered by injection, oxytetracycline was chosen. Research was conducted on two groups of animals: group 1, 100% treated; and group 2, 50% treated and 50% untreated. Oxytetracycline was assayed by a high-performance liquid chromatography-tandem mass spectrometry method. The antibiotic was detectable 2 h post administration in group 1 and group 2 at the concentrations of 10653 ± 1421 μg/kg and 7457 ± 1145 μg/kg, respectively. At withdrawal period (21st day), oxytetracycline concentrations in oral fluid (30.8 ± 9.4 μg/kg in group 1 and 11.6 ± 5.6 μg/kg in group 2) were similar to those determined in muscle (34.5 ± 8.2 μg/kg). The concentrations of oxytetracycline in liver and kidney were 76.8 ± 22 μg/kg and 204 ± 49 μg/kg, respectively. The results of this study indicate that oral fluid analysis can be used for antemortem oxytetracycline detection in pigs, even if the half of animals in one pen are treated.

Intensive and massive pig production often contributes to excessive administration of antibacterials in veterinary medicine. The misuse and failures to follow the label directions of antibiotics, as well as withdrawal inadequacy, can lead to their residue occurrence in products of animal origin. The residue of drugs may result in many biological adverse effects and allergic reactions in consumers as well as the spread of drug-resistant bacteria and bacterial resistance acquisition. Consolidation of pig production requires ensuring adequate conditions of the maintenance of animals with high health conditions, consistent with the guidelines for animal welfare. At the same time, the control of antibiotics in animals is an important element providing the high quality of pigs breeding, as well as the protection of consumers. In residue monitoring programs, the primary material for the control of antibiotics presence in pigs are tissue samples, collected from animals at the slaughterhouse. However, post-mortem analysis does not give the opportunity of monitoring the usage of antibiotics on the farm during the breeding of animals. In ante mortem drug analysis blood can be used as a diagnostic biological matrix; however, blood sampling is connected with certain disadvantages for the animals and the staff responsible for material collection. The post-mortem detection and presence of antibiotic residues in pork tissues above MRL values very often lead to the recall and destruction of significant quantities of meat, which may contribute to economic losses. Therefore, a strong need to find and implement new methods for ante-mortem detection of antibiotics in animals exists, which minimize interference in animal welfare. The results of preliminary studies demonstrate that oral fluid seems to be an effective tool for monitoring the reasonable treatment and residue avoidance. It offers a cost-effective approach for the screening of large populations of animals. The method of oral fluid collection with cotton rope usage allows for the rapid sampling of material. In case of positive oral fluid ante-mortem analysis results, the slaughtering of animals can be delayed, which can bring the avoidance of costs connected with the non-compliant pig meat destruction.

During the 2000s, the problem of pesticide poisoning of honeybees seemed to be almost solved. The number of cases has decreased in comparison to the 1970s. The problem of acute honeybee poisoning, however, has not disappeared, but instead has transformed into a problem of poisoning from ‘traditional’ pesticides like organophosphorus pesticides or pyrethroids, to poisoning from additional sources of ‘modern’ systemic neonicotinoids and fipronil. In this article, the biological activity of pesticides was reviewed. The poisoning symptoms, incident definitions, and monitoring systems, as well as the interpretation of the analytical results, were also reviewed. The range of pesticides, and the detected concentrations of pesticides in poisoned honeybee samples, were reviewed. And, for the first time, cases of poisoning related to neonicotinoids were reviewed. The latter especially is of practical importance and could be helpful to analysts and investigators of honeybee poisoning incidents. It is assumed that secondary poisoning induced by plant collected materials contaminated with systemic pesticides occurs. Food stored in a hive and contaminated with systemic pesticides consumed continuously by the same generation of winter bees, may result in sub-lethal intoxication. This leads to abnormal behaviour identified during acute intoxication. The final result is that the bees discontinue their social role in the honeybee colony super organism, and colony collapse disorder (CCD) takes place. The process described above refers primarily to robust and strong colonies that were able to collect plenty of food due to effective plant protection.