Evaluating the persistence of malachite green residues in tilapia and pacu fish.

Although banned in food-producing animals, residues of malachite green (MG) and its primary metabolite, leucomalachite green (LMG), have been found in fish due to illegal use in aquaculture and the release of industrial wastewater, which represent a serious risk to food and environmental securities. This study aimed to investigate the residue depletion profile of MG and LMG in edible tissues of Nile tilapia (Oreochromis niloticus) and pacu (Piaractus mesopotamicus) cultured simultaneously under the same environmental conditions to support control measures in case of abuse. An analytical method involving QuEChERS sample preparation and liquid chromatography coupled to tandem mass spectrometry was developed, validated, and applied to quantify MG and LMG residues in fish fillets from two depletion experiments after treatment by immersion bath (MG at 0.10 mg L-1 for 60 min). During the experiment, the average water temperature was 30 ºC, while the pH was 6.9. The method is selective, precise (CV = 0.4 - 22%) and accurate (recovery 92 - 114%). The limits of detection and quantification are 0.15 and 0.5 ng g-1, respectively. In both species, the sum of MG and LMG residues were quantified up to the 32nd day post-exposure, and the concentrations were significantly higher in the pacu fillets (up to 3284 ng g-1) than in Nile tilapia (up to 432 ng g-1). The sums of MG and LMG residues were below 2 ng g-1 at 44 days and 342 days for Nile tilapia and pacu, respectively - the Minimum Required Performance Limit (MRPL) for analytical methods intended to monitor forbidden substances in food according to old European Commission guidelines. The persistence of MG residues in pacu may be attributed to its higher lipid content, which favors the accumulation of the non-polar metabolite LMG. These results provide insights into the concern about human, animal, and environmental health risks resulting from unauthorized use or aquatic contamination by industrial wastewater containing MG residues.

Determination of Multi-Class Antimicrobial Residues and Antimicrobial Resistance in Cow Milk and Feces Samples during Withdrawal Period.

The use of antimicrobials in livestock production and their effect on the development of antimicrobial resistance (AMR) is a global health problem for humans, animals and the environment. The aim of this study was to determine antimicrobial residue levels in milk and feces samples during the withdrawal period in dairy cattle administrated with a single dose of the drug, as well as to characterize the antimicrobial resistance patterns of Escherichia coli cultured from feces samples. In the study, dairy cows from three different farms in North Macedonia were included. Raw milk and feces samples were collected before drug administration (0 day) and on the 1st, 2nd, 3rd, 7th and 21st day after drug administration. The antimicrobial residues of oxytetracycline, enrofloxacin, amoxicillin, trimethoprim and procaine-benzylpenicillin were determined using a validated liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) method involving stable isotopes. According to results obtained, the highest levels of analyzed antimicrobial residues were determined on the first day after drug administration, which then gradually decreased until their elimination (7th day). The highest AMR of E. coli (100%) was found in ß-lactam antimicrobials. Less exposure to broad-spectrum antimicrobials could be an important factor for reduction of AMR on dairy farms.

Levamisole incorporation in fish feed - Ensuring the medication dose and avoidance of leaching into the water.

Levamisole, an anthelmintic and immunostimulant drug, has been studied as a promising alternative for aquaculture use. While oral administration through feeding is the main route of administration in fish farming, no studies evaluating methods of levamisole incorporation into the feed have been reported so far. Therefore, this study aimed to evaluate potential procedures for levamisole incorporation in extruded fish feed using ethyl cellulose, gelatin, or vegetable oil, to avoid drug leaching to the water during the animal's medication. A suitable LC-MS/MS method was optimized (full factorial design), validated, and applied to evaluate the efficiency of the process, the homogeneity of the drug concentration, and the leaching rate. The method has been demonstrated to be selective, precise (RSD < 4.9%), accurate (recovery > 98.4%), and linear (r > 0.99, 125-750 mg kg-1). The incorporation procedures using the three coating agents showed high incorporation efficiency (70%) and a homogeneous drug concentration among the extruded feed pellets. A low levamisole leaching rate was verified in the feed prepared using the ethyl cellulose coating procedure (4.3% after 15 min of immersion in the water). On the other hand, fish feed coated with gelatin and oil resulted in a high leaching rate (30-35% after 15 min). Thus, this study shows that coating ethyl cellulose may be a promising procedure for levamisole incorporation in fish feed and with the potential to enhance its use in animal production while reducing environmental contamination.

Multi-class/residue method for determination of veterinary drug residues, mycotoxins and pesticide in urine using LC-MS/MS technique.

BACKGROUND: Veterinary drugs are widely used in animals to prevent diseases and are a complex set of drugs with very different chemical properties. Multiclass and multi-residue methods for simultaneous detection of residues from veterinary drugs and contaminants in urine are very rare or non-existent. Therefore, the aim of this study was to develop and validate a sensitive and reliable quantitative LC-MS/MS method for simultaneous determination of a wide range of veterinary drug and pesticide residues and mycotoxins in bovine urine. This involved 42 veterinary drug residues (4 thyreostats, 6 anabolic hormones, 2 lactones, 10 beta agonists, 15 antibiotics, 5 sulphonamides), 28 pesticides and 2 mycotoxins. Stable isotopically labelled internal standards were used to facilitate effective quantification of the analytes. Analysis was performed in both positive and negative ionization modes with multiple reaction monitoring transitions over a period of 12 min. RESULTS: The parameters validated included linearity, limit of detection (LOD), limit of quantification (LOQ), detection capability (CCß), decision limit (CCα), stability, accuracy and precision. The process followed guidelines of the regulation 2021/808/EC. The calibration curves were linear with coefficient of correlation (R2) from 0.991 to 0.999. The LODs were from 0.01 to 2.71 µg/L, while the LOQs were from 0.05 to 7.52 µg/L. The CCα and CCß were in range 0.05-12.11 µg/L and 0.08-15.16 µg/L. In addition, the average recoveries of the spiked urine samples were from 71.0 to 117.0% and coefficient of variation (CV) < 21.38% (intraday and interday). CONCLUSION: A new isotopic LC-MS/MS method has been developed, validated and applied for identification and quantification of 72 residues of veterinary drugs and pesticides and other contaminants such as mycotoxins in bovine urine. The most appropriated sample preparation procedures involved sodium acetate buffer, enzymatic hydrolysis using ß-glucuronidase and cleanup solid phase extraction with OASIS SPE cartridges. The parameters were satisfactorily validated fulfilling requirements under Regulation 2021/808/EC. Consequently, the method could be used in routine analysis of bovine urine samples for simultaneous detection of veterinary drug and pesticide residues as well as contaminants such as mycotoxins.

Residue Depletion Profile and Estimation of Withdrawal Period for Sulfadimethoxine and Ormetoprim in Edible Tissues of Nile Tilapia (Oreochromis sp.) on Medicated Feed.

Sulfadimethoxine (SDM) and ormetoprim (OMP) are antimicrobials used in combination to treat bacterial infections in fish farming. The use of this drug combination is not yet regulated in some countries, such as Brazil. Due to the lack of regulated drugs for aquaculture in Brazil, this study investigated the residue depletion profile of SDM and OMP in Nile tilapia (Oreochromis sp.) after oral administration. Fish were treated with medicated feed containing a 5:1 ratio of SDM:OMP at the dose of 50 mg kg BW-1 for five consecutive days with an average water temperature of 28 °C. The drugs were incorporated into the feed by using a gelatin coating process which promoted homogeneity in drug concentration and prevented the drug leaching into the water during medication. The SDM and OMP determination in fish fillets (muscle plus skin in natural proportions) was performed using the QuEChERS approach followed by LC-MS/MS quantification. The analytical method was validated according to Brazilian and selected international guidelines. A withdrawal period of 9 days (or 252 °C days) was estimated for the sum of SDM and OMP residues at concentration levels below the maximum residue level of 100 µg kg-1.

Screening for antimicrobial residues in poultry eggs in Bangladesh using Charm II radio-receptor assay technique following validation.

The aim of the study was to screen for the presence of antimicrobial residues in poultry eggs from Bangladesh using the Charm II radio-receptor assay in the absence of expensive confirmatory instrumentation. This was based on cut-off values as set in the validation guidelines according to Commission Decision 2002/657/EC and Commission Implementing Regulation (EU) 2021/808. Fortified eggs spiked with fixed concentrations of doxycycline, erythromycin A, sulphamethazine, and benzylpenicillin were used to determine the cut-off values and detection capabilities (CCß). Other validation parameters included were applicability, ruggedness, and robustness. A total of 201 egg mix samples from native organic chicken, duck, and commercial farm-raised laying hens (both brown and white eggs) were tested and after analysis 13%, 10%, and 4.5% of the egg mix samples showed positive signals for sulphonamides, macrolides/lincosamides, and tetracyclines, respectively. Presence of multiple drug residues were also suspected in 11 out of 201 egg mix samples.

Secondary Validation of a Liquid Scintillation Counter-method for Residues of Tetracyclines, Beta-lactams, and Sulfonamides in Seafood/Aquaculture Products.

This study was carried out to validate the liquid scintillation counter method (Charm II) for the detection of tetracyclines, beta-lactams, and sulfonamides (Sulfa drugs) in a range of Aquaculture Products. This method of validation followed primary validation performed in Belgium and was therefore transferred to Nigeria but further validation was required, and this was performed according to the European Commission Decision 2002/657/EC. Method performance was based on the detection capability (CCß), specificity (cross-reactivity), robustness, repeatability, and reproducibility for the detection of antimicrobial residues. Seafood and aquaculture samples used for the validation process included tilapia (Oreochromis niloctus), catfish (Siluriformes), African threadfin (Galeoides decadactylus), common carp (Cyprinus carpio), and shrimps (penaeidae). These were spiked with varying concentrations of tetracyclines, beta-lactams, and sulfonamides standards to determine the validation parameters. Results of the validation showed tetracyclines had detection capabilities of 50 µg/kg, while beta-lactams and sulphonamides had detection capabilities of 25 µg/kg. The relative standard deviation for both repeatability and reproducibility studies ranged between 1.36% and 10.50%. Results of this study are suitable and comparable to the initial validation reports from the primary validation ofCharm II tests forthedetection ofantimicrobial residues inarange ofaquaculture fish conducted in Belgium. The results also prove the specificity, ruggedness, and reliability of the radio receptor assay tests for detection of the various antimicrobials in aquaculture products. This could be used in seafood/aquaculture products monitoring in Nigeria.

Determination of Veterinary Drug Residues, Mycotoxins, and Pesticide Residues in Bovine Milk by Liquid Chromatography Electrospray Ionisation -tandem Mass Spectrometry.

Introduction: Multi-class and multi-residue analyses are very complex procedures because of the physico-chemical properties of veterinary drug residues and other contaminants. The purpose of the study was to develop an analytical method for the sensitive determination of 69 analytes in bovine milk by liquid chromatography electrospray ionisation-tandem mass spectrometry. Material and Methods: Antimicrobial, anabolic hormone, lactone, ß-agonist, mycotoxin and pesticide residues were analysed in 120 raw milk samples from different dairy farms in North Macedonia. Stable isotopically labelled internal standards were used to facilitate effective quantification of the analytes. Results: The linear regression coefficients were higher than 0.99, the limits of detection ranged from 0.0036 to 47.94 µg/L, and the limits of quantification ranged from 0.053 to 59.43 µg/L. The decision limit values ranged from 0.062 to 211.32 µg/L and the detection capability from 0.080 to 233.71 µg/L. Average recoveries of the analytes spiked in raw milk were in the range of 70.83% to 109%, intra-day coefficient of variation (CV) values from 2.41% to 22.29%, and inter-day CV values from 3.48% to 23.91%. The method was successfully applied in the testing of bovine milk samples. In five samples residues were detected. They were sulfadimethoxine (in two samples), enrofloxacin, tetracycline and oxytetracycline and were at concentrations below the EU maximum residue limit. Conclusion: The method is useful for routine testing for this group of chemical hazards in bovine milk.

A new LC-MS/MS method for multiple residues/contaminants in bovine meat.

A multi-class and multi-residue/contaminant method for the determination of veterinary drug and pesticide residues and mycotoxins in bovine meat has been developed and validated. The veterinary drug residues/contaminants included antimicrobials, anabolic hormones, lactones, ß-agonists, mycotoxins, and pesticides. Isotopic labeled internal standards were included to compensate residual matrix effects. The calibrators used in the method demonstrated linearity with the R2 > 0.98. The decision limit (CCα) values were in the range from 0.067 to 2103.84 µg/kg, while the range for detection capability (CCß) was from 0.083 to 2482.13 µg/kg. The limit of detection (LOD) and limit of quantification (LOQ) were in the range from 0.059 to 291.36 µg/kg, and 0.081 to 328.13 µg/kg, respectively. The recovery of analytes ranged from 61.28% to 116.20%. The intra-day coefficient of variation (CV) was from 0.97 to 25.93% and the inter-day CV was 2.30-34.04%. The method has been used for the determination of 49 residues/contaminants in bovine meat. Application of the method in routine analysis in bovine samples, revealed in limited samples the presences of enrofloxacin, oxytetracycline and sulfadiazine at the concentration of 35.22 µg/kg, 27.35 µg/kg, and 36.20 µg/kg, respectively.

Use of sulfonamides in layers in Kampala district, Uganda and sulfonamide residues in commercial eggs.

BACKGROUND: Use of antimicrobials like sulfonamides in production of layers is a public health risk since it inevitably results in sulfonamide residues in eggs. The presence of the residues may be influenced by knowledge, attitudes and practices of farmers regarding use of sulfonamides (and other antimicrobials) in poultry. OBJECTIVE: The study aimed at assessing the possible contribution of the knowledge, attitudes and practices of poultry farmers to the presence/levels of sulfonamide residues in hen eggs. METHODS: A descriptive cross sectional study was done in the 5 political divisions (and surroundings) of Kampala district. Sixty farmers were systematically sampled from a list of poultry farmers in Kampala and a semi-structured questionnaire administered. Each farmer provided sixty eggs for analysis of sulfadiazine and sulfamethazine residues. Whole eggs were homogenized in acetonitrile and centrifuged twice, extracts evaporated and residues dissolved in mobile phase (32:68, methanol: potassium di-hydrogen phosphate). Fats were removed using hexane while anhydrous sodium chloride was added to break emulsions. Extracts were analyzed by reverse phase high performance liquid chromatography with photodiode array detector. RESULTS: Ninety-five percent of the farmers never observed withdrawal periods although 80% of them knew the importance of withdrawal periods. However, farmers noted that they play a great role in ensuring a safe food supply. Most farmers attributed the non-observance of withdrawal periods to poverty and fear to lose their investments. Ninety-eight percent of the samples had detectable levels of the sulfonamides. Meanwhile, 98.3% of the samples that had detectable sulfonamide residues came from farmers who applied antimicrobials in feeds/ water. CONCLUSION: Consumers of hen eggs in Kampala district are at high risk of sulfonamide residue exposure due to poor farming/ regulatory practices.