Exposure assessment of anticoagulant rodenticides in the liver of red foxes (Vulpes vulpes) in Slovenia.

The study deals with the environmental residues of anticoagulant rodenticides (ARs) in Slovenia to evaluate the toxicological risk of secondary poisoning of red foxes (Vulpes vulpes) as representatives of non-target wildlife, and in relation to the investigated use patterns of ARs and specific local parameters in Slovenia. From 2019 to 2022, 148 liver tissue samples of adult red foxes were collected from almost all state geographical regions. The samples were extracted with methanol/water (2:1, v/v), cleaned-up using a solid supported liquid-liquid extraction, and measured by liquid chromatography-electrospray tandem mass spectrometry (LC-ESI-MS/MS) with reporting limits of 0.5 to 5.0 ng/g. Residues of at least one rodenticide were detected in 77.7 % of the samples. The second generation ARs of bromadiolone, brodifacoum and difenacoum were the most frequently found, appearing in 75.0, 51.4, and 18.9 % of the samples, respectively. Concentrations of pooled ARs ranged from 1.5 to 2866.5 ng/g with mean and median values of 601.4 and 350.2 ng/g, respectively. We determined bromadiolone and brodifacoum at concentrations of ≥800 ng/g in 10.8 and 10.1 % of the samples, and 1.4 and 0.7 % of the samples contained residues >2000 ng/g, respectively. These concentrations are much higher than those found in comparable studies in Europe and elsewhere in the world. Residues of ARs were detected in all monitored statistical regions of Slovenia, with higher concentrations in the eastern parts of the country. First generation ARs were found in only 9.5 % of samples, and residues were below 10 ng/g with one exception (coumatetralyl with 55 ng/g). The results of the study indicate a serious toxicological risk for red foxes in Slovenia as part of the Western Balkans, and will contribute to the growing body of knowledge about the protection of European ecosystems, as wildlife is not limited by national borders.

Experimental Exposure to Bisphenol A Has Minimal Effects on Bone Tissue in Growing Rams-A Preliminary Study.

Bisphenol A (BPA) is a well-known synthetic compound that belongs to the group of endocrine-disrupting chemicals. Although bone tissue is a target for these compounds, studies on BPA-related effects on bone morphology in farm animals are limited. In this preliminary study, we investigated the effects of short-term dietary BPA exposure on femoral morphology, metabolism, mineral content, and biomechanical behavior in rams aged 9-12 months. Fourteen rams of the Istrian Pramenka breed were randomly divided into a BPA group and a control group (seven rams/group) and exposed to 25 µg BPA/kg bw for 64 days in feed. Blood was collected for determination of bone turnover markers (procollagen N-terminal propeptide, C-terminal telopeptide), and femurs were assessed via computed tomography, histomorphometry, three-point bending test, and mineral analysis. BPA had no significant effects on most of the parameters studied. Only mineral analysis showed decreased manganese (50%; p ≤ 0.05) and increased copper content (25%; p ≤ 0.05) in the femurs of BPA-exposed rams. These results suggest that a 2-month, low-dose exposure to BPA in growing rams did not affect the histomorphology, metabolism, and biomechanical behavior of femurs; however, it affected the composition of microelements, which could affect the histometric and biophysical properties of bone in the long term.

Bisphenol A in edible tissues of rams exposed to repeated low-level dietary dose by high-performance liquid chromatography with fluorescence detection.

The presented work deals with levels and distribution of bisphenol A (BPA) in the edible tissues of a large food-producing animal species. An experimental animal study included 14 young Istrian pramenka rams (Ovis aries), of which seven were exposed for 64 days to a low dietary dose of BPA at 25 µg/kg b.w./day, and seven served as a control group. Residue analysis of both aglycone and total BPA was performed in the muscle tissue, liver, kidney and fat tissue of the individual animals by means of enzymatic deconjugation (for total BPA), organic solvent extraction, molecularly imprinted polymer solid-phase extraction (MISPE) clean-up and high-performance liquid chromatography with fluorescence detection (HPLC-FLU). The analysis was optimized and validated for aglycone BPA in the fat tissue and for the total BPA in all tissues investigated. Edible tissues of the control group of rams generally remained BPA-free, while there were concentration differences between the control and treated groups for liver and kidney post last administration. The human health risk resulting from this study was assessed by the estimated dietary exposure in adults, which was < 0.1% related to the valid European Union Tolerable Daily Intake (TDI) value of 4 µg/kg b.w./day. However, it would be 58-fold higher than the newly proposed TDI value of 0.04 ng/kg b.w./day.

Basic Exploratory Study of Bisphenol A (BPA) Dietary Administration to Istrian Pramenka Rams and Male Toxicity Investigation.

Bisphenol A (BPA), an endocrine-disrupting chemical and environmental pollutant, has been reported by many researchers to induce male reproductive toxicity in different experimental models. In this study, we investigated whether long-term exposure for two months to 25 µg/kg body weight (low dose) of BPA affects spermatogenesis or sperm quality in young Istrian Pramenka rams exposed via diet. We evaluated body and testicular weights, histopathology of testes and epididymides, and sperm analyses, and compared these parameters between the group of treated rams and the control group of rams. Although there were some differences between the two groups, these differences were not large or statistically significant. The only statistically significant difference was the lower epithelial height of seminiferous tubules in treated rams, compared to control rams. In addition to assessing toxicity, BPA concentrations in the blood plasma of treated rams were determined after the first administration, and the toxicokinetic parameters of total BPA were calculated. In this study, no major signs of altered reproduction in rams were detected.

Seminiferous epithelium cycle staging based on the development of the acrosome in ram testis.

Testicular histopathology is considered the most sensitive and reliable method to detect the effects of chemicals on sperm production. To carry out a sensitive examination of testicular histopathology and interpret the changes require knowledge of spermatogenic stages. Spermatogenic staging based on acrosome development during spermiogenesis is conventionally performed in animal species routinely used for research and toxicity testing. In contrast, small ruminants, such as sheep and goats, are rarely used as animal models to evaluate toxicity in male reproductive organs. To the best of our knowledge, a comparable spermatogenic staging system in rams has not yet been fully characterised. Hence, this study aimed to adapt the existing spermatogenic staging based on acrosome development in bull testes to fit the seminiferous epithelium cycle of ram testes. The results show that spermatogenic staging based on acrosome development in bull testes can, with slight modifications, be efficiently used for the staging of ram testes.

A rapid analysis of Bisphenol A using MISPE coupled with HPLC-FLD in tissues of food-producing animals.

Bisphenol A (BPA) is a highly-produced organic compound of anthropogenic origin with a wide-range use and is ubiquitously present in both living organisms and the environment. A previously published analytical method for testing of the free (aglycone) BPA in foodstuffs was simplified and optimized for sheep muscle tissue, kidney and liver, by using only a single MISPE purification step allied with HPLC-FLD analysis. The recovery rates and RSD values over a concentration range of 1-10 µg/kg were in the range of 67‒86% and 3‒34%, respectively, while linearity in the matrix, represented by the r2, was ≥0.999. LOD values were 0.5‒1 µg/kg and LOQ values were 1 µg/kg. The analytical method used is a contribution to the field of veterinary toxicology and food testing and also proved to be applicable for other food-producing animal species, e.g. pigs, poultry and freshwater fish. The MISPE sorbent material for testing of BPA was reusable, with up to five re-use cycles without significant loss in performance characteristics.•The paper reports on a rapid determination of free (aglycone) BPA in tissues of food-producing animals by HPLC-FLD and presents a substantial improvement of analytical performance with regard to time- and cost-savings, as well as environmental protection.•MISPE is an advanced analytical technology, and the results proved that in a single and simplified SPE step it had good performance characteristics such as selectivity, recovery and precision, allied with low LOD and LOQ values.•It was proved that MISPE BPA cartridges could be used at least five times without significant loss of methodological recovery and precision.

Sudden outbreak of metastatic intestinal adenocarcinoma in rainbow trout Oncorhynchus mykiss.

Intestinal adenocarcinomas are uncommon in fishes. To date, they have been reported in zebrafish Danio rerio, blue gularis Fundulopanchax sjostedti, koi carp Cyprinus carpio koi, Atlantic salmon Salmo salar and rainbow trout Oncorhynchus mykiss. Metastases are even rarer and have been observed so far at very low prevalence, only in feed-induced adenocarcinoma in Atlantic salmon and rainbow trout. Intestinal adenocarcinoma with liver and heart metastases and mesenteric invasion was found in approximately 33% of 4 yr old rainbow trout from a Slovene hatchery with 2000 breeding trout. During stripping, lumps in the abdominal cavity were palpated in one-third of the breeding fish; some of the fish were anorectic and lethargic, and mortality was slightly increased. Affected trout were euthanized and 4 were submitted for necropsy and histopathology. Necropsy revealed firm, whitish, irregularly lobular masses originating from the intestine. Histologically, the intestinal masses showed a prominent proliferation of tall columnar neoplastic epithelial cells arranged in dense irregular islands or solid areas and papillotubular protuberances. Solid areas of neoplastic cells were also observed in the mesentery of all trout and in the liver of one trout, whereas minute groups of neoplastic cells were seen in the vessels of the intestinal mucosa in all trout and in the myocardium and the liver of one trout. Epithelial origin of neoplastic cells was confirmed by expression of the cytokeratin marker AE1/AE3. The intestinal masses were diagnosed as intestinal adenocarcinoma with mesenteric invasion and metastases to the liver and heart. The cause of intestinal adenocarcinoma was not determined.

Determination of free and total bisphenol A in the urine and feces of orally and subcutaneously dosed sheep by high-performance liquid chromatography with fluorescence detection.

An analytical procedure has been introduced to enable a study of the excretion of free bisphenol A (BPA), total BPA and its main metabolite bisphenol A glucuronide (BPA-GLUC). In the experiment, in which 100 µg/kg b. w. BPA was administered daily to one Istrian Pramenka sheep for 5 days with consecutive urine and feces samples being taken, BPA and total BPA were determined in samples using high-performance liquid chromatography (HPLC) with fluorescence detection. Because of their good recovery, precision, and sensitivity, the methods have also proved applicable to further ecotoxicological studies of free BPA, BPA-GLUC and total BPA. The results were subsequently compared with reported field studies of BPA in livestock excreta.

Preliminary toxicokinetic study of BPA in lactating dairy sheep after repeated dietary and subcutaneous administration.

Dietary intake is the predominant route of human exposure to bisphenol A and one of the important food commodities is milk. The aim of our study was to preliminarily evaluate the bisphenol A exposure and disposition in sheep milk after repeated dietary and subcutaneous administration of a relatively low dose (100 µg/kg of b. w./day) of bisphenol A to a sheep. On the basis of blood plasma sampling, milk sampling and HPLC analysis, we developed the toxicokinetic model. With the toxicokinetic model we showed that most likely only free bisphenol A passes into the mammary gland and is subsequently conjugated there. The percentage of the dose eliminated with milk was less than 0.1%, regardless of the route of bisphenol A administration. It is proven that the bisphenol A is eliminated through the milk of lactating sheep. However, the amounts excreted in the milk that were detected in this study are minimal.

Analysis and Occurrence of Bisphenol A in Mediterranean Mussels (Mytilus galloprovincialis) Sampled from the Slovenian Coastal Waters of the North Adriatic Sea.

During 2015, a selection of Mediterranean mussels (Mytilus galloprovincialis) and seawater were sampled along the Slovenian coast, which is part of the North Adriatic Sea, in order to test for the presence of bisphenol A (BPA). The concentration of the BPA in the tissues of mussels sampled from the farms and open sea was from < 0.03 to 0.55 µg kg-1 of w.w. and from < 0.03 to 0.46 µg kg-1 of w.w., respectively. The mussel shells from the farms and open sea contained from 0.01 to 0.3 µg BPA kg-1 of w.w. and from 0.04 to 0.27 µg BPA kg-1 of w.w., respectively. In a harbour, mussel tissue and shell concentration of BPA was 0.21 and 0.18 µg BPA kg-1 of w.w., respectively. The seawater in the shellfish farms and the open sea was contaminated with an average of 0.007 µg l-1 of BPA.

Recent developments in the analysis of avermectin and milbemycin residues in food safety and the environment.

A review of the developments on the analysis of residues of avermectins and milbemycins (both macrocyclic lactones) is presented. The macrocyclic lactones (MLs) are an important class of chemicals, which are used worldwide as veterinary drugs and as crop protection agents. As a result, residues of MLs are important from both a food safety and environmental perspective. A review of the developments in ML residues in food was carried out in detail in 2006. As a result, this paper covers recent developments in the area of food analysis, which are mainly multi-residue assays based on liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). A brief coverage of HPLC fluorescence (HPLC-FLD) based methods is included for completeness. The paper will carry out a comprehensive review of ML residues in environmental samples. These additional sections are reflective of the growing number of research papers published on LC-MS/MS and environmental applications in recent years.

Influence of P-glycoprotein inhibition on secretion of ivermectin and doramectin by milk in lactating sheep.

The aim of to the present study was to evaluate the effects of verapamil (VER) on plasma pharmacokinetics of ivermectin (IVM) and doramectin (DOR) in lactating Istrian Pramenka dairy sheep and to investigate the role of P-glycoprotein (P-gp) in transport of avermectins into milk. Pharmacokinetics of IVM and DOR following subcutaneous administration of 0.2mg/kg b.w. was evaluated in four groups of sheep. They were administered either IVM or DOR alone or in combination with verapamil (VER) at a dose of 3.0mg/kg b.w., 3 times at 12h intervals. Blood plasma and milk samples were collected at defined time intervals over 30 days post-treatment to determine IVM and DOR concentration levels. Pharmacokinetic parameters in sheep injected with IVM or DOR alone corresponded to previously published values. Comparison between sheep injected with IVM only, and sheep injected with IVM in combination with VER (IVM+VER) showed significant difference in pharmacokinetic parameters in blood plasma. Area under the concentration-time curve (AUC) truncated at 2 days (AUC(2)) was 15 and 28 µg day/L for group IVM and IVM+VER, respectively. With co-administration of VER, apparent plasma clearance (Cl/F) and mean residence time (MRT) of IVM decreased from 135 to 116 L/day and from 5.8 to 3.8 days, respectively. Similar trends were observed for DOR (AUC(2) 48 vs. 68 µg day/L, Cl/F 61 vs. 46 L/day, and MRT 5.6 vs. 4.4 days for groups DOR and DOR+VER, respectively). This study confirms that co-administration of VER has a significant effect on pharmacokinetic parameters of subcutaneously administered IVM in blood plasma. The influence on DOR pharmacokinetics is much weaker. This could be either due to the difference in lipophilicity or the difference in affinity towards P-gp as a result of structural differences. No significant influence of VER on AUC ratio of IVM and DOR between milk and plasma was observed suggesting that P-gp does not govern transport of avermectins into milk.

Trace analysis of endectocides in milk by high performance liquid chromatography with fluorescence detection.

An analytical method has been developed for the simultaneous determination of the following endectocide drugs in milk: ivermectin, abamectin, doramectin, moxidectin, eprinomectin, emamectin and nemadectin. Samples were extracted with acetonitrile, purified with solid-phase extraction on a reversed phase C(8), derivatised with N-methylimidazole, trifluoroacetic anhydride and acetic acid to a stable fluorescent derivative, and were further analysed by gradient high performance liquid chromatography (HPLC) on an endcapped reversed phase Supelcosil LC-8-DB. The derivatisation step was mathematically optimised and the method was validated according to the requirements of Commission Decision 2002/657/EC, using fortified raw bovine milk. Mean recovery was between 78 and 98%. The repeatability (CV(r)) and within-laboratory reproducibility (CV(W)) ranged from 4.6 to 13.4% and from 6.6 to 14.5%, respectively. Decision limits (CCalpha) for analytes with MRL values, namely eprinomectin and moxidectin, were determined to be 24.8 and 50.6 microg kg(-1), respectively. CCalpha values for unauthorised endectocides ranged from 0.1 to 0.2 microg kg(-1). Due to high acceptability regarding the required criteria and applicability to ovine and caprine milk, giving similar results, this multi-analyte method has been successfully implemented in pharmacokinetic research studies as well as statutory residue monitoring in Slovenia.

Linearity of eprinomectin pharmacokinetics in lactating dairy sheep following pour-on administration: excretion in milk and exposure of suckling lambs.

Pharmacokinetics of eprinomectin (EPR) were studied in blood plasma and milk in two groups of six Istrian Pramenka dairy sheep and their suckling lambs following pour-on administration of EPR to ewes at dose levels of 0.5 and 1mg/kg. Maximum concentration in plasma was 2.22 and 5.25 microg/l, and AUC was 13.6 and 33.7 microg day/l for the 0.5 and 1.0mg/kg dose, respectively. These results indicate that drug exposure with a dose of 0.5mg/kg, which is commonly used in cattle, may be subtherapeutic. The concentration time course in milk paralleled plasma concentrations. In the dose range studied, linear pharmacokinetics of EPR were demonstrated. Milk-to-plasma AUC ratio was 0.79+/-0.12 and 1.12+/-0.43; the fraction of dose recovered in milk was 0.037+/-0.011 and 0.058+/-0.027% for the low and high dose, respectively. Maximum residual levels in milk were below the maximum acceptable level of 20 microg/kg; however, EPR was detected in all samples investigated. Despite low permeability in milk, AUC in plasma of suckling lambs was between 20 and 30% of the AUC in plasma of ewes.

Kinetics of abamectin disposition in blood plasma and milk of lactating dairy sheep and suckling lambs.

Abamectin (ABM) has been used worldwide as an anthelmintic drug in veterinary medicine and as an agricultural pesticide. Its pharmacokinetics and permeation into milk was evaluated in dairy sheep after subcutaneous administration. ABM elimination half-lives and mean residence times were 1.7 and 3.7 days for blood plasma and 1.9 and 3.8 days for milk, respectively. The ABM milk to plasma concentration ratio (0.89) primarily depends on milk fat content. Transfer of ABM residues to suckling lambs was evaluated by determination of ABM concentration time courses in lambs' plasma. Mean maximal concentration in lambs was 1.6 microg L(-1) at 3.3 days, and elimination half-life was 2.7 days. In ewes' plasma and milk, ABM was detected up to 23 days. Because of different pharmacokinetics, ABM exposure in lambs was almost 10% of the exposure in ewes, although the amount excreted in milk was only 1.0% of the dose.

Determination of residues of azaperone in the kidneys by liquid chromatography with fluorescence detection.

An analytical method has been developed for the quantitative determination of residues of the tranquillizer azaperone (AZN) in the kidneys of slaughtered animals. Samples were extracted with acetonitrile, extracts were acidified and further purified with solid-phase extraction (SPE) on a polymeric mixed-mode cation-exchange sorbent, Oasis. AZN and its main metabolite azaperol (AZL) were eluted by alkaline methanol (MeOH), the eluate was evaporated, re-dissolved and analysed by gradient high performance liquid chromatography (LC) on reversed and deactivated phase LiChrospher 60-RP select B at excitation and emission wavelengths of 245 and 345 nm, respectively. The method was validated according to the requirements of European Commission Decision 2002/657/EC, using fortified porcine kidneys. The method proved to be selective, specific against carazolol (CAR) and linear over a concentration range 10-150 microg kg(-1) (r2>0.99). Over a concentration range 50-150 microg kg(-1), mean recovery of AZN and AZL was 88.2 and 91.2%, respectively, with intra-laboratory reproducibility of <11.0 and <9.0%, respectively. The decision limit (CCalpha) of AZN and AZL was 112 and 111 microg kg(-1), respectively, and the limit of quantification (LOQ) was 10 and 5 microg kg(-1), respectively. The procedure was also applied to bovine, poultry and horse kidneys, giving similar results, and has been successfully implemented in statutory residue monitoring control in food of animal origin in Slovenia.

Analytical procedure for determination of the time profile of eprinomectin excretion in sheep faeces.

An analytical procedure has been introduced to enable study of the time profile of eprinomectin excretion in sheep faeces. Eprinomectin was extracted from sheep faeces with acetonitrile, the extract was cleaned by solid-phase extraction (SPE), and, after derivatization by reaction with N-methylimidazole, trifluoroacetic anhydride, and acetic acid, eprinomectin was analysed by high-performance liquid chromatography (HPLC) with fluorescence detection. The method has a low detection limit (1.0 ng g(-1) of moist sheep faeces), a low quantification limit (2.5 ng g(-1) of moist sheep faeces), good recovery (in the range 78.8 to 87.1%), and good reproducibility (RSD<10%). The method was used to study the time-profile of excretion of eprinomectin in sheep faeces after a single topical administration of 0.5 mg kg(-1) b.w. of the drug. Because of its good recovery, precision, and sensitivity, the method has also proved applicable to further ecotoxicological studies of eprinomectin.

Review of methodology for the determination of macrocyclic lactone residues in biological matrices.

The macrocyclic lactones (MLs) are probably the anti-parasitic agents most widely used in the treatment of food producing animals, poultry, aquaculture and crops. Ivermectin was the first macrocyclic lactone product to be licensed for use about 20 years ago. A number of alternative products such abamectin, doramectin, emamectin, eprinomectin, moxidectin, milbemycin and selamectin, have been marketed since. Because of the increase in the number of ML drugs, there has been a steady increase in the number of published analytical methods for determination of their residues. In this paper, the structure and properties of the different ML drugs available on the market are described. The occurrence and persistence of ML residues in food is discussed in relation to marker residues and current maximum residue limits (MRLs) as defined in the European Union (EU). Methodologies for determination of ML residues in biological matrices are described in terms of extraction and clean-up methods used for different matrices. Detection systems for determination of ML residues are discussed with a particular emphasis placed on new developments in screening technologies and liquid chromatography with fluorescence or mass spectrometry.

Performance characteristics of an analytical procedure for determining chloramphenicol residues in muscle tissue by gas chromatography-electron capture detection.

Validation of an analytical method for determining chloramphenicol residues in muscle tissue by gas chromatography-electron capture detection (GC-ECD) was performed according to the latest European Union criteria for the analysis of veterinary drugs in food, laid down by Commission Decision 2002/657/EC. The method using the meta isomer of chloramphenicol as an internal standard proved to be very selective, specific to other related phenicols and accurate to within +3.6% at a concentration level of 8.9 microg/kg, as present in the certified reference material available. The correlation coefficient of the calibration curve was 0.9991. At all three fortification levels studied (0.3, 0.45 and 0.6 microg/kg), repeatability and intra-laboratory reproducibility were <8 and < or =9%, respectively. The decision limit (CCalpha) and detection capability (CCbeta) were 0.07 and 0.12 microg/kg, respectively. The validation results and the results of participation in an international inter-laboratory proficiency test indicate that the method presented is completely suited for regulatory control to screen and quantify chloramphenicol residues in various muscle tissues on a routine basis.

Abamectin effects on rainbow trout (Oncorhynchus mykiss).

The effect of abamectin (ABM) on rainbow trout (Oncorhynchus mykiss) was studied. The acute toxicity of ABM on rainbow trout was established, following the target 58-h water bath exposure of ABM concentrations from 0.6 to 4.5 microg/l, on the basis of which LD75 (4.0 microg/l) was calculated. The histological changes in organs showed a direct toxicity of ABM for rainbow trout since degenerative changes in brain and kidney and--to a minor extent--in liver were established. The values of the ABM residues in fish muscle tissue with skin were proportional to the exposed concentrations of ABM.