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.

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.

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.

Combination of extractive techniques followed by HPLC-MS/MS analysis to monitor ent-agathic acid in fish treated with Copaifera duckei Dwyer.

Plants are used as therapeutic alternatives in Veterinary Medicine, including therapies for food-producing animals. However, these medicinal resources can sometimes contain dangerous substances, and when used in animals that supply food, they stand out from the point of view of food safety. The diterpene ent-agathic acid, a component of Copaifera duckei oleoresin, is an example of substances already described with toxic activity in mammals. Thus, this study aimed to propose combining two extractive techniques followed by high-performance liquid chromatography coupled mass spectrometry analysis to monitor residues of ent-agathic acid in Piaractus mesopotamicus fillet treated in an immersion bath with Copaifera duckei oleoresin. An optimized combination of solid-liquid extraction (using acidified acetonitrile) and dispersive liquid-liquid microextraction (using acidified water and chloroform as dispersive and extracting solvent, respectively) was performed to recover the target analyte, added to the development of HPLC-MS/MS method with adequate validation parameters to quantify the ent-agathic acid present in the fish fillet. In vivo tests of residual persistence of ent-agathic acid in fishes treated with C. duckei oleoresin were performed, indicating the non-detection of the target diterpene (< 6.1 µg/mL). The combined extractive procedure followed by quantitative analysis in the in vivo test of residual persistence of the target analyte in fish indicated the absence of ent-agathic acid in all samples. Thus, the data found might contribute to understanding the use of oleoresins from C. duckei as an alternative to traditional veterinary products.

The association of modified QuEChERS and DLLME to offer high analytical detectability to assess residual depletion profile of erythromycin in fish.

An analytical method for the determination of erythromycin A (ERY) residues in fish fillet was developed, optimized, and validated employing a modified QuEChERS procedure associated to DLLME technique as a preconcentration step. The obtained LOD and the LOQ were 0.1 µg kg-1 and 1 µg kg-1, respectively. The validated method provides linearity in the range of 1 to 20 µg kg-1, precision (CV < 6.3 %) and accuracy (recovery ranging from 103 to 110 %). The procedure was applied in an experimental study to evaluate the residual depletion profile of ERY in fish (Piaractus mesopotamicus) after oral administration. The treatment was carried out at a daily dose of 100 mg (kg BW)-1 of ERY, for 7 consecutive days and with an average water temperature of 30 °C. A withdrawal time of 240°-day was estimated for eliminating ERY residues at concentration levels below the maximum residue limit considered (MRL 100 µg kg-1).

Food safety evaluation for the use of albendazole in fish: residual depletion profile and withdrawal period estimation.

Due to the lack of drugs regulated for aquaculture, we have evaluated the use of albendazole (ABZ) - a potential drug to be regulated for fish - under food safety perspectives assessing the depletion profile of ABZ and its main metabolites (albendazole sulphoxide - ABZSO, albendazole sulphone - ABZSO2 and albendazole amino sulphone - ABZ-2-NH2SO2) in fish fillets (muscle and skin) after single dose oral administration of 10 mg ABZ kg-1 body weight. For the drug administration, a suitable procedure for ABZ incorporation into fish feed was employed, obtaining good homogeneity of ABZ concentration among feed pellets (CV<4.1%) and low drug leaching when medicated feed remained in the water for up to 60 min (<2.7%). After medication, fish were euthanised at 8, 12, 24, 48, 72, 96 and 120 h and fillets collected. Depletion studies in various fish species (patinga and tilapia) were conducted simultaneously, under water temperature at 30.4 ± 0.3 °C and pH 6.8 ± 0.1. The highest concentrations for the sum of residues (ABZ, ABZSO, ABZSO2 and ABZ-2-NH2SO2) in fish fillet were 1210 ng g-1 in patinga and 637 ng g-1 in tilapia. Under the employed rearing conditions, the obtained results did not indicate a requirement for a minimum withdrawal period to be proposed for tilapia considering the maximum residue limit of 100 µg g-1, since the determined residual concentration was

Emamectin benzoate in tilapia: Alternative method for drug incorporation into feed and associated residual depletion study.

Due to the lack of regulated drugs for aquaculture, the present study considered specific issues relating to environmental and food safety aspects concerning the potential use of emamectin benzoate (EMA) in freshwater fishes such as tilapia (Oreochromis niloticus) - an important commercial fish species worldwide. The residual depletion of EMA (EMA-B1a) in fillet (muscle plus skin in natural proportions) of tilapia treated with a daily dose of 50 µg/kg BW during seven consecutive days was evaluated. To facilitate this, analytical methods for quantitation of EMA in fish feed and in fish fillet employing LC-MS/MS were developed and validated. To eliminate the risk of EMA leaching from feed into the aquatic environment during fish medication via oral administration, a promising procedure for drug incorporation into feed involving the coating of feed pellets with ethyl cellulose polymer containing EMA was evaluated. The medicated feed exhibited good homogeneity (CV < 2.1%) with negligible EMA release (< LOQ) when the medicated feed remained in the water for up to 20 min. Depletion study analysis revealed the highest EMA concentration obtained in fish fillet to be 13.3 ng/g. Therefore, under the employed rearing conditions of this study, the obtained results did not evidence requirement for a minimum withdrawal period to be proposed considering the maximum residue limit of 100 µg/g for fish muscle. In response to the well-recognized demands and need for new alternative veterinary drugs for use within aquaculture, this study offers impetus for consideration of EMA use in tilapia taking into account environmental contamination and food safety issues.

LC-MS/MS methods for albendazole analysis in feed and its metabolite residues in fish fillet and a leaching study in feed after an alternative procedure for drug incorporation.

This paper describes the development of analytical methods for the quantification of albendazole (ABZ) in fish feed and ABZ and its main known metabolites (albendazole sulfoxide, albendazole sulfone and albendazole aminosulfone) in fish fillet employing LC-MS/MS. In order to assess the reliability of the analytical methods, evaluation was undertaken as recommended by related guides proposed by the Brazilian Ministry of Agriculture for analytical method validation. The calibration curve for ABZ quantification in feed showed adequate linearity (r > 0.99), precision (CV < 1.03%) and trueness ranging from 99% to 101%. The method for ABZ residues in fish fillet involving the QuEChERS technique for sample extraction had adequate linearity (r > 0.99) for all analytes, precision (CV < 13%) and trueness around 100%, with CCα < 122 ng g-1 and CCß < 145 ng g-1. Besides, by aiming to avoid the risk of ABZ leaching from feed into the aquatic environment during fish medication via the oral route, a promising procedure for drug incorporation in the feed involving coating feed pellets with ethyl cellulose polymer containing ABZ was also evaluated. The medicated feed had good homogeneity (CV < 3%) and a lower release of ABZ (< 0.2%) from feed to water when the medicated feed stayed in the water for up to 15 min.

LC-MS/MS methods for sulfadimethoxine and ormetoprim analysis in feed and fish fillet and a leaching study for feed after alternative procedures for the incorporation of drugs.

This paper describes the method development for sulfadimethoxine (SDM) and ormetoprim (OMP) quantitation in fish feed and fish fillet employing LC-MS/MS. In order to assess the reliability of the analytical method, valuation was undertaken as recommended by guidelines proposed by the Brazilian Ministry of Agriculture. The calibration curve for the quantification of both drugs in feed showed adequate linearity (r > 0.99), precision (CV < 12%) and trueness ranging from 97% to 100%. The method for the determination of SDM and OMP residues in fish fillet involved a simple sample preparation procedure that had adequate linearity (r > 0.99), precision (CV < 16%) and trueness around 100%, with CCα < 100.2 ng g-1 and CCß < 100.4 ng g-1. With a goal of avoiding the risk of drug leaching from feed into the aquatic environment during fish medication via the oral route, different procedures for drug incorporation into feed were evaluated. Coating feed pellets with ethyl cellulose polymer containing the drug showed promising results. In this case, medicated feed released drugs to water at a level below 6% when the medicated feed stayed in the water for up to 15 min.